Association of adiponectin and its receptor gene polymorphisms with the risk of coronary heart disease in northern Guangxi.

OBJECTIVE: To investigate the association of circulating adiponectin (APN) level and single nucleotide polymorphisms (rs1501299 and rs266729) of the APN gene in the coronary heart disease (CHD) population of Northern Guangxi Province. METHODS: Two hundred and sixty-three CHD patients and 235 healthy controls from our hospital from August 2018 to October 2020 were included in this study. ELISA was used to determine the serum APN concentration. PCR-RFLP and direct DNA sequencing were used to analyze the genotypes of APN gene rs1501299 G/T and rs266729 C/G single-nucleotide loci, their distribution differences between the two groups were compared and their correlation with APN concentration was analyzed. RESULTS: The serum APN concentration in the CHD group was significantly lower than the control group (14.40(1.42-52.26) µg/mL vs. 29.40 (3.18-90.31) µg/mL, P < 0.001). There were statistically significant differences in the rs266729 genotype of APN single nucleotide locus between the two groups (P < 0.001). The dominant model and recessive model of rs266729 genotype showed that mutant homozygous GG genotype carriers significantly increased the risk of CHD in comparison with C allele carriers (CG + CC) (OR = 2.156, 95 %CI: 1.004-4.631, P = 0.049), and this effect was still significant after adjusting gender and age (OR = 2.695, 95 %CI 1.110-6.540, P = 0.028). In both the dominant and recessive models for rs1501299, ORs before and after adjustment for age and sex revealed no significant association with CHD, with ORs of 0.765 (95 % CI: 0.537-1.091, P = 0.139) and 0.718 (95 % CI: 0.466-1.106, P = 0.133) in the Dominant model, and ORs of 0.960 (95 % CI: 0.442-2.087, P = 0.918) and 0.613 (95 % CI: 0.239-1.570, P = 0.308) in the Recessive model, respectively. No statistically significant differences in APN concentrations across genotypes in both groups (P > 0.05), with chi-square values of 1.633 (control group) and 0.823 (CHD group) for rs1501299, and 1.354 (control group) and 0.618 (CHD group) for rs266729. CONCLUSIONS: APN gene of rs266729 C/G single-nucleotide loci gene mutation can significantly increase the risk of CHD. There was no significant correlation between rs1501299 G/T single-nucleotide loci and CHD in Northern Guangxi populations.

Tobacco transcription factor bHLH123 improves salt tolerance by activating NADPH oxidase NtRbohE expression.

In plants, reactive oxygen species (ROS) produced following the expression of the respiratory burst oxidase homolog (Rboh) gene are important regulators of stress responses. However, little is known about how plants acclimate to salt stress through the Rboh-derived ROS signaling pathway. Here, we showed that a 400-bp fragment of the tobacco (Nicotiana tabacum) NtRbohE promoter played a critical role in the salt response. Using yeast one-hybrid (Y1H) screens, NtbHLH123, a bHLH transcription factor, was identified as an upstream partner of the NtRbohE promoter. These interactions were confirmed by Y1H, electrophoretic mobility assay, and chromatin immunoprecipitation assays. Overexpression of NtbHLH123 resulted in greater resistance to salt stress, while NtbHLH123-silenced plants had reduced resistance to salt stress. We also found that NtbHLH123 positively regulates the expression of NtRbohE and ROS production soon after salt stress treatment. Moreover, knockout of NtRbohE in the 35S::NtbHLH123 background resulted in reduced expression of ROS-scavenging and salt stress-related genes and salt tolerance, suggesting that NtbHLH123-regulated salt tolerance is dependent on the NtbHLH123-NtRbohE signaling pathway. Our data show that NtbHLH123 is a positive regulator and acts as a molecular switch to control a Rboh-dependent mechanism in response to salt stress in plants.

A novel bHLH transcription factor, NtbHLH1, modulates iron homeostasis in tobacco (Nicotiana tabacum L.).

Iron (Fe) is a major micronutrient which influences plant growth, development, quality and yield. Although basic helix-loop-helix (bHLH) transcription factors (TFs) which respond to iron deficiency have been identified, the molecular mechanisms have not been fully elucidated. In this study, a novel bHLH TF, NtbHLH1, was found to be induced by iron deficiency. Further analysis indicated that NtbHLH1 is localized to the nucleus and functions as a transcriptional activator. Moreover, overexpression of NtbHLH1 resulted in longer roots, altered rhizosphere pH and increased ferric-chelate reductase activity in iron deficient conditions. Overall these changes resulted in increased iron uptake relative to wild type plants. NtbHLH1 mutants, on the other hand, had an opposite phenotype. In addition, transcript levels of seven genes associated with iron deficiency response were higher in the NtbHLH1 overexpression transgenic plants and lower in ntbhlh1 relative to the WT under iron deficiency treatment. Taken together, these results demonstrated that NtbHLH1 plays a key role in iron deficiency response and they provide new insights into the molecular basis of iron homeostasis in tobacco.

The AP2 transcription factor NtERF172 confers drought resistance by modifying NtCAT.

Drought stress often limits plant growth and global crop yields. Catalase (CAT)-mediated hydrogen peroxide (H2 O2 ) scavenging plays an important role in the adaptation of plant stress responses, but the transcriptional regulation of the CAT gene in response to drought stress is not well understood. Here, we isolated an APETALA2/ETHYLENE-RESPONSIVE FACTOR (AP2/ERF) domain-containing transcription factor (TF), NtERF172, which was strongly induced by drought, abscisic acid (ABA) and H2 O2 , from tobacco (Nicotiana tabacum) by yeast one-hybrid screening. NtERF172 localized to the nucleus and acted as a transcriptional activator. Chromatin immunoprecipitation, yeast one-hybrid assays, electrophoretic mobility shift assays and transient expression analysis assays showed that NtERF172 directly bound to the promoter region of the NtCAT gene and positively regulated its expression. Transgenic plants overexpressing NtERF172 displayed enhanced tolerance to drought stress, whereas suppression of NtERF172 decreased drought tolerance. Under drought stress conditions, the NtERF172-overexpressed lines showed higher catalase activity and lower accumulation of H2 O2 compared with wild-type (WT) plants, while the NtERF172-silenced plants showed the inverse correlation. Exogenous application of amino-1,2,4-triazole (3-AT), an irreversible CAT inhibitor, to the NtERF172-overexpression lines showed decreased catalase activity and drought tolerance, and increased levels of cellular H2 O2 . Knockdown of NtCAT in the NtERF172-overexpression lines displayed a more drought stress-sensitive phenotype than NtERF172-overexpression lines. We propose that NtERF172 acts as a positive factor in drought stress tolerance, at least in part through the regulation of CAT-mediated H2 O2 homeostasis.

Genome-wide identification and expression analysis of the WRKY gene family in common tobacco (Nicotiana tabacum L.).

The coding products of WRKY gene family plays important roles in plant growth and development as well as in various stress responses. They have been identified in various plants, but only few in common tobacco (Nicotiana tabacum L.). In this study, 164 putative WRKY proteins in the common tobacco genome were identified by using the conserved WRKY sequence (PF03106) from the Pfam database. Phylogenetic trees, functional domain analysis, chromosomal localization, subcellular localization and tissue expression patterns were analyzed with the bioinformatics softwares, including DNAMAN 5.0, Weblogo 3, MEGA 5.1, MG2C and MEME. First of all, phylogenetic trees divided all the candidate genes into three subfamilies: Ⅰ, Ⅱ and Ⅲ, respectively, and subfamily Ⅱ could be further divided into five subgroups: group Ⅱ-a, -b, -c, -d and -e. Secondly, the WRKY regions contained a highly conserved heptapeptide stretch WRKYGQK followed by a zinc-finger motif. Most of the NtWRKY genes contained 2-5 exons and a highly conserved gene structure. Thirdly, 154 out of 164 NtWRKY genes were distributed with different densities on 24 chromosomes, and each subfamily with different patterns and frequency. The largest number of NtWRKY genes was found on chromosome VI, and only one on chromosome X. Fourthly, the majority of NtWRKY members located in the nucleus, with 74 percent of subfamily Ⅲ in the extracellular matrix. Lastly, the members in the same subfamily had different spatial and temporal expression profiles, with 11 NtWRKY genes in roots, stems and leaves expressed at various levels. The expression of genes NtWRKY26, NtWRKY30 and NtWRKY32 can be induced by Phytophthora nicotianae. Our research thus provides valuable information for NtWRKY gene cloning and functional characterization in common tobacco.

[Analysis of Gene Deficiency Types of Thalassemia in Lingui District of Guilin City].

OBJECTIVE: To analyze the gene defect types and distribution characteristics of α- and ß-thalassemia in Lingui District of Guilin City, Guangxi, so as to provide scientific basis for genetic consultation and prevention measures. METHODS: A total of 6 496 suspected cases for screening the thalassemia during physical examination, premarital examination, pregnancy examination and hospitalization in the Second Affiliated Hospital of Guilin Medical University from May 2016 to October 2019 were analyzed. Gap-PCR, PCR-RDB and DNA sequencing techniques were used to detect the types and constituent ratios of gene defects in α- and ß-thalassemia positive cases. RESULTS: Among 6 496 suspected patients, 1 363 were thalassemia carriers, the total positive rate was 20.98%. There were 677 cases of single-gene deletion and 26 cases of double-gene detetion on the deletional α-thalassemia, 115 cases of non-deletion α-thalassemia mutation and 4 cases of deletion plus mutation. The positive rate of α-thalassemia was 12.66%. There were 11 gene abnormalities for α-thalassemia, of which --SEA/αα (50.36%) was the most common, followed by -α3.7/αα (23.84%); the main α-gene mutation was ααCS (6.93%). There were 514 ß-thalassemia gene carriers, with a positive rate of 7.93%. In 12 types of ß-gene mutations, CD41-42 (-TTCT) (55.64%) was the most common, followed by CD17 (A→T) (20.23%). There were 25 cases of double heterozygous α and ß thalassemia (0.39%), of which -α3.7/ßCD17 (24%) and --SEA/ß41-42 (16%) were numerically dominant. Two of rare thalassemia genotypes were identified by sequencing, which were heterozygous mutations of Chinese Hong Kong type α thalassemia (HKαα/αα or HKαα/-α3.7) and ß gene mutations IVS-I (-2) or codon30 (A→G) ß0, respectively. CONCLUSION: Lingui district of Guilin city is a high incidence area of thalassemia. The mutation rate of α-thalassemia --SEA/αα type deletion is relatively high, followed by that of the right deletion type (-α3.7/αα). CD41-42 (-TTCT) has the highest mutation rate in ß-thalassemia, followed by CD17(A→T). The results of this study provide reference data for the regional screening, diagnosis and treatment of thalassemia and eugenics.

Construction of a SNP Fingerprinting Database and Population Genetic Analysis of Cigar Tobacco Germplasm Resources in China.

Cigar tobacco is an important economic crop that is widely grown around the world. In recent years, varietal identification has become a frequent problem in germplasm preservation collections, which causes considerable inconvenience and uncertainty in the cataloging and preservation of cigar germplasm resources, in the selection of parental lines for breeding, and in the promotion and use of high quality varieties. Therefore, the use of DNA fingerprints to achieve rapid and accurate identification of varieties can play an important role in germplasm identification and property rights disputes. In this study, we used genotyping-by-sequencing (GBS) on 113 cigar tobacco accessions to develop SNP markers. After filtering, 580,942 high-quality SNPs were obtained. We used the 580,942 SNPs to perform principal component analysis (PCA), population structure analysis, and neighbor joining (NJ) cluster analysis on the 113 cigar tobacco accessions. The results showed that the accessions were not completely classified based on their geographical origins, and the genetic backgrounds of these cigar resources are complex and diverse. We further selected from these high-quality SNPs to obtained 163 SNP sites, 133 of which were successfully converted into KASP markers. Finally, 47 core KASP markers and 24 candidate core markers were developed. Using the core markers, we performed variety identification and fingerprinting in 216 cigar germplasm accessions. The results of SNP fingerprinting, 2D barcoding, and genetic analysis of cigar tobacco germplasm in this study provide a scientific basis for screening and identifying high-quality cigar tobacco germplasm, mining important genes, and broadening the basis of cigar tobacco genetics and subsequent breeding work at the molecular level.

Construction of arming Yarrowia lipolytica surface-displaying soybean seed coat peroxidase for use as whole-cell biocatalyst.

Whole-cell biocatalysts could be used in wide-ranging applications. In this study, a new kind of whole-cell biocatalyst was successfully constructed by genetically immobilizing soybean seed coat peroxidase (SBP) on the cell surface of Yarrowia lipolytica Po1h, using a new integrative surface display expression vector (pMIZY05). The coding sequence of SBP was optimized and chemically synthesized, then inserted into pMIZY05 to generate expression plasmid pMIZY05-oEp. A DNA fragment corresponding to SBP and selection marker expression cassettes, without bacterial sequences, was released from pMIZY05-oEp by enzyme digestion and used to transform host yeast cells. A transformant (CM11) with a high recombinant SBP activity of 1571.9 U/mL was obtained, and recombinant SBP was proved to be successfully anchored on cell surface by testing the activities of different cellular fractions. After optimization of culture conditions, the recombinant SBP activity of CM11 was increased to 4187.8 U/mL. Afterwards, biochemical properties of the recombinant SBP were determined: optimum catalytic conditions were 37.5℃ at pH 3.5, and recombinant SBP exhibited high stability during thermal or acidic treatment. Recombinant activity of cell-displayed SBP was re-examined at optimum temperature and pH, which promoted an increase up to 4432.5 U/mL. To our knowledge, this represents the highest activity ever reported for heterologous expression of SBP. This study also provides a useful strategy for heterologous expression of proteins which could be toxic to intracellular content of host cells.

Tobacco Transcription Factor NtbHLH123 Confers Tolerance to Cold Stress by Regulating the NtCBF Pathway and Reactive Oxygen Species Homeostasis.

Cold stress is a major environmental factor that impairs plant growth and development, geographic distribution, and crop productivity. The C-repeat binding factor (CBF) regulatory pathway has an essential role in response to cold stress. Here, we characterized a bHLH transcription factor from Nicotiana tabacum, NtbHLH123, in response to cold stress (4°C). Overexpression of NtbHLH123 enhanced cold tolerance in transgenic tobacco plants. Based on yeast one-hybrid, chromatin immunoprecipitation PCR, and transient expression analysis assays, NtbHLH123 binds directly to the G-box/E-box motifs in the promoter of the NtCBF genes and positively regulates their expression. Furthermore, NtbHLH123-overexpressing plants showed lower electrolyte leakage, reduced malondialdehyde contents, H2O2 and reactive oxygen species (ROS) accumulation under cold stress, which contributed to alleviating oxidative damage to the cell membrane after cold stress treatment. And NtbHLH123 increased stress tolerance by improving the expression of a number of abiotic stress-responsive genes to mediate the ROS scavenging ability and other stress tolerance pathways. Taken together, we present a model suggesting that NtbHLH123 is a transcriptional activator that functions as a positive regulator of cold tolerance by activating NtCBF, ROS scavenging-related, and stress-responsive genes.