Application value of triglyceride-glucose index and triglyceride-glucose body mass index in evaluating the degree of hepatic steatosis in non-alcoholic fatty liver disease.

BACKGROUND: The elevation of TyG is considered an important factor in promoting the progression of non-alcoholic fatty liver disease (NAFLD), but its impact on the degree of liver steatosis remains unclear. This study aims to explore the relationship between TyG and TyG-related indices, such as triglyceride glucose-body mass index (TyG-BMI), with the degree of liver fat accumulation. METHODS: From January 2021 to March 2022, 1171 participants underwent health check-ups, and all underwent FibroScan transient elastography. The analysis focused on identifying the factors that contribute to the onset of NAFLD and the degree of hepatic steatosis. RESULTS: The predictive value of TyG-BMI (OR = 1.039, 95% CI 1.031-1.046) in triggering NAFLD development was greater than that of TyG alone. The areas under the curve for TyG-BMI and TyG were calculated at 0.808 and 0.720, respectively. TyG-BMI (OR = 1.034, P < 0.001) was identified as a main independent factor affecting hepatic steatosis severity. With each incremental increase in TyG-BMI, the likelihood of experiencing an increase in the extent of hepatic steatosis was 1.034 times higher than that of the preceding unit. CONCLUSIONS: The TyG-BMI showed higher accuracy in predicting NAFLD than did the TyG, and was more closely linked to the severity of hepatic steatosis. Therefore, it can be included as a parameter in health management centers and should be widely used to screen and evaluate patients with NAFLD.

Series-temporal transcriptome profiling of cotton reveals the response mechanism of phosphatidylinositol signaling system in the early stage of drought stress.

Plants are sessile organisms suffering severe environmental conditions. Drought stress is one of the major environmental issues that affect plant growth and productivity. Although complex regulatory gene networks of plants under drought stress have been analyzed extensively, the response mechanism in the early stage of drought stress is still rarely mentioned. Here, we performed transcriptome analyses on cotton samples treated for a short time (10 min, 30 min, 60 min, 180 min) using 10% PEG, which is used to simulate drought stress. The analysis of differently expressed genes (DEGs) showed that the number of DEGs in roots was obviously more than that in stems and leaves at the four time points and maintained >2000 FDEGs (DEGs appearing for the first time) from 10 min, indicating that root tissues of plants respond to drought stress quickly and continuously strongly. Gene ontology (GO) analysis showed that DEGs in roots were mainly enriched in protein modification and microtubule-based process. DEGs were found significantly enriched in phosphatidylinositol signaling system at 10 min through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, implying the great importance of phosphatidylinositol signal in the early stage of drought stress. What was more, two co-expression modules, which were significantly positively correlated with drought stress, were found by Weighted Gene Co-expression Network Analysis (WGCNA). From one of the co-expression modules, we identified a hub-gene Gohir.A07G058200, which is annotated as "phosphatidylinositol 3- and 4-kinase" in phosphatidylinositol signaling system, and found this gene may interact with auxin-responsive protein. This result suggested that Gohir.A07G058200 may be involved in the crosstalk of phosphatidylinositol signal and auxin signal in the early stage of drought stress. In summary, through transcriptome sequencing, we found that phosphatidylinositol signaling system is an important signal transduction pathway in early stage in response to drought stress, and it may interact with auxin signal transduction through phosphatidylinositol 3- and 4-kinase.

Effect and mechanism of vitamin D on the development of colorectal cancer based on intestinal flora disorder.

BACKGROUND: To investigate the correlation between the level of circulating vitamin D and the development of colorectal cancer (CRC) and to clarify the effect and mechanism of vitamin D on the development of CRC. METHODS: Serum samples from 63 patients with CRC (CRC group) and 61 healthy volunteers (normal group) were collected. Azoxymethane + dextran sodium sulfate-induced CRC mouse model and dietary models with different doses of vitamin D were established to verify whether vitamin D supplementation could reverse the occurrence and development of CRC at the overall animal level. Intestinal barrier integrity and microbial defense response were evaluated by detection of intestinal flora and expression of related genes. RESULTS: In the clinical serum samples, compared with the normal group, the level of 25 (OH) D3 in the CRC group was relatively low (P < 0.01), which was consistent with the clinical situation in mice. Vitamin D deficiency aggravated the deterioration of enteritis and intestinal cancer in CRC mice, whereas the overall condition of CRC mice improved after vitamin D supplementation. Vitamin D has a significant regulatory effect on the homeostasis of the intestinal flora, particularly in the regulation of intestinal probiotics, Akkermansia muciniphila-mediated colon barrier integrity. CONCLUSIONS: Vitamin D deficiency is closely related to the high incidence of CRC, and vitamin D supplementation can inhibit the occurrence and development of CRC. Vitamin D plays a role in the reversal of CRC mainly through the regulation of intestinal flora, especially the regulation of A. muciniphila-mediated colon barrier integrity.

Enhanced platelet function through CAR-T cell therapy in relapsed/refractory multiple myeloma.

The influence of chimeric antigen receptor T (CAR-T) cell therapy on platelet function in relapsed/refractory (R/R) multiple myeloma (MM) has not been thoroughly investigated. Our cohort comprised fifty MM patients treated with CAR-T cells. The mean platelet closure time (PCT) induced by collagen/adenosine diphosphate (CADP) in peripheral blood was significantly prolonged before lymphodepletion (195.24 ± 11.740 s) and notably reduced post-CAR-T cell therapy (128.02 ± 5.60 s), with a statistically significant improvement (67.22, 95% CI 46.91-87.53, P < 0.001). This post-treatment PCT was not significantly different from that of healthy controls (10.64, 95% CI 1.11-22.40, P > 0.05). Furthermore, a pronounced enhancement in PCT was observed in patients with a response greater than partial remission (PR) following CAR-T cell infusion compared to pre-treatment values (P < 0.001). An extended PCT was also associated with a less favorable remission status. In patients with cytokine release syndrome (CRS) grades 0-2, those with a PCT over 240.5 s exhibited a shorter progression-free survival (PFS), with median PFS times of 10.2 months for the PCT > 240.5 s group versus 22.0 months for the PCT ≤ 240.5 s group. Multivariate analysis revealed that a PCT value exceeding 240.5 s is an independent prognostic factor for overall survival (OS) in R/R MM patients after CAR-T cell therapy. The study demonstrates that CAR-T cell therapy enhances platelet function in R/R MM patients, and PCT emerges as a potential prognostic biomarker for the efficacy of CAR-T cell therapy.

Association between triglyceride glucose-related markers and the risk of metabolic-associated fatty liver disease: a cross-sectional study in healthy Chinese participants.

OBJECTIVES: This study aimed to evaluate the performance of the triglyceride glucose (TyG) index and its related markers in predicting metabolic-associated fatty liver disease (MAFLD) in healthy Chinese participants. DESIGN: This was a cross-sectional study. SETTING: The study was conducted at Health Management Department of the Affiliated Hospital of Xuzhou Medical University. PARTICIPANTS: A total of 20 922 asymptomatic Chinese participants (56% men) were enrolled. OUTCOME MEASURES: Hepatic ultrasonography was performed to diagnose MAFLD based on the latest diagnostic criteria. The TyG, TyG-body mass (TyG-BMI) and TyG-waist circumference indices were calculated and analysed. RESULTS: Compared with the lowest quartile of the TyG-BMI, the adjusted ORs and 95% CIs for MAFLD were 20.76 (14.54 to 29.65), 92.33 (64.61 to 131.95) and 380.87 (263.25 to 551.05) in the second, third and fourth quartiles, respectively. According to the subgroup analysis, the TyG-BMI in the female and the lean groups (BMI<23 kg/m2) showed the strongest predictive value, with optimal cut-off values for MAFLD of 162.05 and 156.31, respectively. The areas under the receiver operating characteristic curves in female and lean groups were 0.933 (95% CI 0.927 to 0.938) and 0.928 (95% CI 0.914 to 0.943), respectively, with 90.7% sensitivity and 81.2% specificity in female participants with MAFLD and 87.2% sensitivity and 87.1% specificity in lean participants with MAFLD. The TyG-BMI index demonstrated superior predictive ability for MAFLD compared with other markers. CONCLUSIONS: The TyG-BMI is an effective, simple and promising tool for predicting MAFLD, especially in lean and female participants.

Sex-specific prevalence and risk factors of metabolic-associated fatty liver disease among 75,570 individuals in eastern China.

Background: Metabolic-associated fatty liver disease (MAFLD) is a newly proposed definition and there is limited data on MAFLD prevalence. We aimed to investigate the prevalence of MAFLD in an eastern Chinese population. Methods: This cross-sectional study included participants from an eastern Chinese population who underwent regular health checkups. Based on current diagnostic criteria, MAFLD was diagnosed in individuals with both hepatic steatosis and metabolic disorders. The overall and stratified prevalence derived based on sex, age, body mass index (BMI), and various metabolic disorders were estimated. Multivariate logistic regression analysis was used to determine the risk factors for MAFLD. Results: Among the 75,570 participants, the overall prevalence of MAFLD was 37.32%, with higher rates in men (45.66%) than in women (23.91%). MAFLD prevalence was highest in men aged 40-49 years (52.21%) and women aged 70-79 years (44.77%). In all the BMI subgroups, the prevalence was higher in men than in women. In both sexes, the prevalence of MAFLD increased as BMI levels increased. Furthermore, MAFLD was associated with metabolic disorders, especially in the female participants with severe obesity (odds ratio 58.318; 95% confidence interval: 46.978-72.397). Conclusion: MAFLD is prevalent in the general adult population in eastern China. Sex-specific differences in MAFLD prevalence were identified based on age, BMI, and metabolic disorders. MAFLD is associated with metabolic disorders, particularly obesity.

[Antiulcer effects and mechanism study of Veronicastrum axillare on ethanol induced gastric ulcer rats].

OBJECTIVE: To study the antiulcer effects and the mechanism of Veronicastrum axillare (Sieb. et Zucc) Yamazaki (VAY) on ethanol induced gastric ulcer rats. METHODS: Totally 48 healthy SD rats were randomly divided into 6 groups, i.e., the normal group, the model group, the ranitidine group, the high dose VAY group, the medium dose VAY group, and the low dose VAY group, 8 in each group. Rats in the normal group and the model group were administered with normal saline respectively. Rats in the ranitidine group were administered with 0.18% ranitidine suspension (at the daily dose of 0.027 g/kg) by gastrogavage. Those in the high dose VAY group, the medium dose VAY group, and the low dose VAY group were administered with VAY at the daily dose of 2.8 g/kg, 1.4 g/kg, and 0.7 g/kg by gastrogavage, once daily for 14 consecutive days. The gastric ulcer model was established using absolute ethanol after the last gastrogavage. The ulcer index and the ulcer inhibitory rate were compared. The concentrations of malonyldialdehyde (MDA), nitric oxide (NO), epidermal growth factor (EGF), and the activity of superoxide dismutase (SOD) in the serum and the homogenate of the gastric mucosa tissue were detected. RESULTS: Compared with the model group, the gastric ulcer index in the rest groups obviously decreased (P < 0.01). The ulcer index was dose-dependent with VAY (P < 0.01), with the highest gastric ulcer index shown in the high dose VAY group (P < 0.01). Compared with the normal group, the concentrations of MDA and NO significantly increased in the serum and the gastric mucosa tissue, the activity of SOD and the EGF content in the gastric mucosa tissue of rats in the model group significantly decreased (P < 0.01). Compared with the model group, the MDA concentrations in the serum and the gastric mucosa tissue decreased, the serum NO content increased, the NO content in the gastric mucosa tissue decreased, the serum SOD activity increased, the EGF content in the gastric mucosa tissue increased in the rest groups, all showing statistical difference (P < 0.05, P < 0.01). CONCLUSIONS: The water extract of VAY had significant effects on ethanol induced gastric ulcer. Its mechanisms might lie in reducing the generation of free radicals, promoting the oxygen free radical clearance, restraining lipid peroxidation, regulating and controlling the in vivo contents of NO and EGF.

KLB gene polymorphism is associated with obesity and non-alcoholic fatty liver disease in the Han Chinese.

Klotho beta (KLB) mediates binding of fibroblast growth factor (FGF) 21 to the FGF receptor (FGFR). FGF21-KLB-FGFR signaling regulates multiple metabolic systems in the liver, and we hypothesized that FGF21, KLB and FGFR single-nucleotide polymorphisms (SNPs) are involved in hepatic lipid accumulation. The SNPs were detected in 1688 individuals divided into four groups: non-obese without non-alcoholic fatty liver disease (NAFLD), obese without NAFLD, non-obese with NAFLD, and obese with NAFLD. The A-allele of KLB SNP rs7670903 correlated with higher body mass index (P = 0.0005), and the A-allele frequency was higher in the obese than non-obese group (P = 0.003). The G-allele frequency of KLB rs7674434 and T-allele frequency of rs12152703 were higher in the obese with NAFLD than obese without NAFLD group (P = 0.004 and P = 0.006), but the genotype distribution between two non-obese groups did not differ. KLB rs7674434 and rs12152703 had associations with alanine aminotransferase (ALT) (P = 0.03 and P = 0.04, respectively) and gamma-glutamyltransferase (P = 0.03 and P = 0.02, respectively) levels in all subjects, but the associations were especially strong with ALT in the NAFLD group (P = 0.005 and P = 0.008, respectively). These findings suggest that KLB SNPs are related to obesity and hepatic inflammation and that they may be involved in the pathogenesis of NAFLD.

The expression of classical swine fever virus structural protein E2 gene in tobacco chloroplasts for applying chloroplasts as bioreactors.

It has been reported that genes encoding antigens of bacterial and viral pathogens can be expressed in plants and are shown to induce protection antibodies. The structural protein E2 of classical swine fever virus (CSFV), which has been shown to carry critical epitopes, has been expressed in different systems. Here, we report the expression of CFSV E2 gene in tobacco chloroplasts. Mice immunized with leaf extracts elicited specific antibodies. This indicated that the expressed E2 proteins had a certain degree of immunogenicity. To our knowledge, this is the first report showing induction of protective antibody in response to classical swine fever virus (CSFV) by immunization with antigen protein E2 expressed in tobacco chloroplasts, which will open a new way to protection from CSFV by plant chloroplasts as bioreactors.

Stable expression of foot-and-mouth disease virus protein VP1 fused with cholera toxin B subunit in the potato (Solanum tuberosum).

The expression vector, pBI121CTBVP1, containing the fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene was constructed by fused PCR and transferred into potato (Solanum tuberosum L.) by Agrobacterium-mediated transformation. Transformed plants were obtained by selecting on kanamycin-resistant medium strictly and regenerated. The transgenic plantlets were identified by PCR, Southern-blot and the production of fused protein was confirmed and quantified by Western-blot and ELISA assays. The results showed that the fused genes were expressed stablely under the control of specific-tuber patatin promoter. The expressed fused proteins have a certain degree of immunogenicity.

Recombination and expression of classical swine fever virus (CSFV) structural protein E2 gene in Chlamydomonas reinhardtii chroloplasts.

The expression of classical swine fever virus (CSFV) structural protein E2 in different vectors, which has been shown to carry critical epitopes, has been established. Here, we reported a Chlamydomonas reinhardtii chloroplast expression vector, P64E2, containing classical swine fever virus structural protein E2 gene, which was constructed and transferred to C. reinhardtii by biolistic bombardment method. The transformants were identified by PCR, Southern blotting, Western blotting after selecting on resistant media. ELISA quantification assay showed that the expressed E2 protein accumulated up to 1.5-2% of the total soluble protein. The results of the study on the immunological activity indicated that the protein E2 expressed in C. reinhardtii chloroplasts could elicit animal bodies to produce antibodies against protein E2.

Expression profiles of a cytoplasmic male sterile line of Gossypium harknessii and its fertility restorer and maintainer lines revealed by RNA-Seq.

The Gossypium harknessii background cytoplasmic male sterility (CMS) system has been used in cotton hybrid breeding in China. However, the mechanism underlying pollen abortion and fertility restoration in CMS remains to be determined. In this study, we used RNA-seq to identify critical genes and pathways associated with CMS in G. harknessii based CMS lines (588A), the near isogenic restorer lines (588R), and maintainer lines (588B). We performed an assembly of 80,811,676 raw reads into 89,939 high-quality unigenes with an average length of 698 bp. Among these, 72.62% unigenes were annotated in public protein databases and were classified into functional clusters. In addition, we investigated the changes in expression of genes between 588A and 588B (588R); the RNA-seq data showed 742 differentially expressed genes (DEGs) between 588A and 588B and 748 DEGs between 588A and 588R. They were mainly down-regulated in 588A and most of them distributed in metabolic and biosynthesis of secondary metabolites pathways. Further analysis revealed 23 pollen development related genes were differentially expressed between 588A and 588B. Numerous genes associated with tapetum development were down-regulated in 588A, implicating tapetum dysplasia may be a key reason for pollen abortion in CMS lines. Also, among DEGs between 588A and 588R, we identified two PPR genes which were highly up-regulated in restorer line. This study may provide assistance for detailed molecular analysis and a better understanding of harknessii based CMS in cotton.

Cloning and Expression Analysis of Eight Upland Cotton Pentatricopeptide Repeat Family Genes.

The pentatricopeptide repeat (PPR) gene family is one of the largest gene families in plants. Most PPR genes are localized in mitochondria and chloroplasts functioning in regulation of plant growth and development, fertility restoration for cytoplasmic male sterility (CMS), and stress defense. In this study, using in silico cloning and PCR amplification with degenerate primers based on Arabidopsis PPR genes, we cloned eight new full-length PPR genes encoding protein sequences ranging from 458 to 875 amino acids, with 8 to 16 repetitive PPR elements in upland cotton and all of them lack introns. Expression analysis revealed that eight PPR genes were differently expressed in roots, stems, leaves, and floral buds. As for GhI12, its expression in floral buds at days 3-5 was significantly higher in line 777R (restorer line) than in line 777A (CMS line). Further tests with real-time PCR showed that GhI12 expression peaked at day 3 in 777R, followed by a gradual decline, while its expression fluctuated in 777A, peaking at day 5 and day 13. In addition, Gh155c17 and GhI12 were upregulated under salt stress. This is the first report of upland cotton PPR genes involved in salt stress response.

Molecular phylogeny of the domesticated silkworm, Bombyx mori, based on the sequences of mitochondrial cytochrome b genes.

Pupae from the Chinese wild mulberry silkworm, Bombyx mandarina, and 11 representative strains of the domesticated silkworm, Bombyx mori were selected for preparation of mitochondrial DNA. The 5'-end fragments of cytochrome b genes (Cytb) were generated by polymerase chain reaction products and sequenced directly. The homologous sequences of the Japanese B. mandarina and three strains of B. mori were from the GenBank database. The sequences of the 16 silkworm strains were analysed with DNASTAR software and a phylogenic tree was constructed using PHYLIP software. The result showed that: (i) The sequence divergence between the strains of B. mori and the Japanese B. mandarina was larger (5.4%-5.8%) compared with that between strains of B. mori and the Chinese B. mandarina (0.8% -1.9%). Analysis of clustering also showed that the sequences of B. mori strains and Chinese B. mandarina clustered into group (B group), while that of Japanese B. mandarina (A group) was outside this cluster. This may be evidence for the hypothesis that B. mori originated from Chinese B. mandarina. (ii) Among 14 strains of B. mori, sequence divergence was small and the most divergence was seen between strains Yanhe-1 and Chuxiong, whose sequences branched off from those of the other B. mori strains on the phylogenetic tree. From this and from historical records, we infer that the strains Yanhe-1 and Chuxiong originated independently from southwest China.

A scaleless wings mutant associated with tracheal system developmental deficiency in wing discs in the silkworm, Bombyx mori.

A mutant of Bombyx mori has wings with few scales and is named scaleless. We investigated the morphology of this mutant and found that it had many fewer wing scales than the corresponding wild type (WT) silkworm and that the remaining scales were smaller in shape with fewer furcations. Reciprocal transplantation of wing discs between scaleless and WT revealed that the WT wing disc could develop into a small wing with scales after transplantation into a scaleless larva; however, the scaleless wing disc developed into a small wing without scales in a WT larva. Upon dissection of WT and scaleless wing discs at different stages from the fifth instar larva to adulthood, no obvious differences were found before pupation. However, after pupation, tracheae produced from WT wing veins extended to the lacunae between the veins and formed a network on the second day after pupation, whereas this did not happen in scaleless. At the same time, no marked difference in adult body tracheal development was found between the mutant and wild type. Furthermore, if the surface of a wing disc was cut and its veins injured, the resulting wing also had fewer scales than the corresponding WT. Also, we found that higher partial pressure of O2 could rescue the loss of scales in scaleless. These data suggest that the factors affecting the growth of scales were not produced in the hemolymph, but in the wing disc itself. It is also implied that wing scale development is dependent on the correct organization of the tracheal system in the wing disc.

Transplastomic Plants Homoplasmic for Foreign Transgenes.

Scientists pay more and more attention to the research on plastid engineering for its following advantages foreign genes can be integrated site-specifically into the plastid genome (plastome) there are no position effects as experienced with random insertion of transgenes in nuclear transformation pollen-mediated dispersion of transgenes can be avoided because chloroplasts are maternally transmitted gene silencing does not occur in plastids and therefore transgene expression is stable in progeny of transplastomic plants and the high ploidy level of the plastome in leaf cells makes high levels of transgene expression feasible. At the same time, however, the highly polyploid plastome makes it difficult to get transplastomic plants homoplasmic for foreign transgenes. In this work, chloroplast transforming vector pTRCH205, which carries two psbA5'-nifH-psbA3'and Prrn-aadA-TpsbA cassettes flanked by plastid DNA sequence to target their insertion between psbA and trnK operons, was constructed. Plastid transformation of Nicotiana tabacum was carried out by the biolistic delivery of transforming plasmid pTRCH205 DNA into leaf cells. Integration of nifH and aadA by two homologous recombination events via the flanking ptDNA sequences, and selective amplification of the transplastomes on MS medium with high concentration of spectinomycin, yielded resistant cell lines. All the independent transplastomic lines were subjected to three additional rounds of regeneration and were subcultured for 6--10 times through stem sections on MS medium containing 500 mg/L spectinomycin, to obtain homoplasmic tissues. The results of PCR assay and Southern blot hybridization, probed with 0.9 kb BglII/SnaBI homologous fragment, indicated that foreign genes had been integrated into the plastomes of transgenic plants, which finally became homoplasmic for foreign transgenes.

[Protein splicing and its application in protein engineering].

Protein splicing, which is an intein mediated posttranslational processing, involves a series of intramolecular cleavage-ligation reactions. Intein is an intervening polypeptide which can catalytic self-cleavage from a pre-protein accompanied by the concomitant joining of the two flanking polypeptides (the extein) through a peptide bond. Protein splicing not only enriches genetic theory of posttranslational processing, but also have wide application prospect.

[The insect resistance of OC and Bt transplastomic plant and the phenotype of their progenies].

The Bt gene and OC gene were cotransformed to tobacco chloroplast with particle bombardment method and spectinomycin resistance tobacco seedlings were obtained. Bioassays showed that the transgenic tobacco containing both genes had enhanced toxicity to the larvae of cotton bollworm (helicoverpa zea) by comparison with the plants containing only Bt or OC gene. Southern-blotting analysis and genetic analysis of progenies showed that the Bt and OC gene expressed and was inherited maternally to the progenies.

Survival of human metallothionein-2 transplastomic Chlamydomonas reinhardtii to ultraviolet B exposure.

Solar ultraviolet (UV) radiation has a great influence on green organisms, especially plankton like Chlamydomonas. A human metallothionein-2 gene, which is generally considered to have an anti-radiation function by its coding product, was transferred into the chloroplast genome of Chlamydomonas reinhardtii. To dynamically measure the UV effects on Chlamydomonas cells grown in liquid tris-acetate-phosphate medium, a new method was developed based on the relationship between the chlorophyll content of an algal culture and its absorbance at 570 nm after the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In this experiment, both the wild-type and the transplastomic C. reinhardtii cells were cultivated in 96-well microplates containing liquid tris-acetate-phosphate medium in the absence or presence of zinc, copper, cadmium and cysteine. The transgenic C. reinhardtii showed a higher resistance than wild-type to UV-B exposure under all the examined conditions. Metals in the medium had positive impacts on both types of cells, but had significant influence only on the transplastomic cells. However, the high cell viability of the transgenic alga at the end of the 8 h UV-B treatment disappeared after a 20-h recovery culture. Cysteine did not protect cells from UV-B damage, but clearly enhanced the growth of both wild-type and transgenic C. reinhardtii.

[Expression of the gene coding for a thermostable alpha-amylase from Pyrococcus furious in Chiamydomonas reinhardtii chloroplast ].

Thermostable alpha-amylase from Pyrococcus furious is an important industrial enzyme in brewing and alcohol production. Eexpression of the thermostable a-amylase in plants can reduce greatly costs in the production of alcohol using crop plants. A chloroplast expression vector, p64A, containing the thermostable alpha-amylase gene from Pyrococcus furious, was constructed with clpP-trnL-petB-chlL-rp123-rpl2 as Chlamydomonas reinhardtii plastid homologous recombinant fragments and spetinomycin-resistant aadA gene as select marker. The plasmid p64A was transferred into the chloroplast genome of C. reinhardtii by the biolistic method. Nine independently transformed lines were obtained by 100 mg/L spectinomycin selection. PCR amplification, Southern blot analysis of the transgene and cultivation in the dark all showed that the a-amylase gene had been integrated into chloroplast genome of C. reinhardtii. The activity of amylase expressed in the chloroplast of C. reinhardtii was detected by amylase activity assay and found to be as high as 77.5 u/g fresh weight of cells. These experimental results demonstrated the possibility of using transgenic chloroplasts of plant as bioreactors for production of industrial enzymes.