Current Biotechnology

Progress on the Application of Mitochondrial SSR Molecular Markers in Plants
Lanlan ZHANG, Caihua LI, Yuzhu FANG, Yan SONG, Wanlin KANG, Zhiyu LI, Xiao ZHANG, Rui ZHANG
2023, 13(6): 821-826. DOI: 10.19586/j.2095-2341.2023.0074
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Mitochondrial simple sequence repeat （mtSSR） is a new molecular marker technology developed in recent years. It not only has the advantages of high polymorphism， wide distribution and codominance of SSR markers， but also has the advantages of maternal inheritance and rapid structural evolution of plant mitochondrial genome. It has important applications in population genetic structure， gene flow evolution， species evolution， cytoplasmic genetic characteristics， population classification and other aspects. Based on existing research results both domestically and internationally，the article reviewed the research progress of mtSSR molecular markers on plant population classification and cytoplasm identification， and summarized the plant mtSSR databases，which aimed to provid reference for using mtSSR technology in plant research.

Research Progress on Social Hierarchy of Shrimps and Crabs
Hangyu QI, Tingting DU, Quanxin GAO, Qiongying TANG, Guoliang YANG, Shaokui YI
2023, 13(6): 827-836. DOI: 10.19586/j.2095-2341.2023.0091
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Social hierarchy determines the allocation of resources within an animal population， reduces unnecessary fighting between populations， and plays an important role in the survival and reproduction of animals. Studying the social hierarchy has important theoretical significance and applied values for improving the production of shrimps and crabs in aquaculture， but there have been no in-depth reports yet. This paper summarized the types of social hierarchy of shrimps and crabs， and further reviewed the internal and external factors affecting the formation of social hierarchy and the behavioral changes of shrimps and crabs affected by social hierarchy and the mechanism of pheromone， as well as chemical communication in social behavior. Meanwhile， prospects were made for future research on social hierarchy of shrimps and crabs， aiming to provide a reference for in-depth exploration of the biological traits of shrimps and crabs in aquaculture.

Application of Caenorhabditis elegans Model in the Study of Memory and Forgetting Behavior
Xin ZHAO, Xinyu LI, Minghao LI, Shiyi ZHOU, Yaqi DENG, Zhiyuan ZHENG, Wei ZOU
2023, 13(6): 837-843. DOI: 10.19586/j.2095-2341.2023.0077
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Memory is the basis of learning and mastering new knowledge， and forgetting helps to maintain the efficiency of the brain memory system， so memory and forgetting are important components of the normal operation of the brain neural network. With its small size， short life cycle， and easy recognition of single neurons， Caenorhabditis elegans has become one of the ideal models for neuroscience and behavioral research. Studies based on C. elegans model combined with higher model organisms to explore the mechanisms of memory and forgetting will help reveal the occurrence of diseases related to abnormal memory and forgetting. In this paper， we reviewed the molecular mechanism of memory and forgetting behavior of C. elegans widely used in volatile substances and pathogenic bacteria， and the application of transgenic C. elegans in memory and forgetting related diseases， so as to provide theoretical reference for subsequent research on memory and forgetting.

Research Progress on Secondary Metabolites of Marine Streptomyces
Qiaoli CHEN, Jie HUANG, Senyu CHEN, Shaoting PAN, Lingzhi TANG, Xuan HONG
2023, 13(6): 844-852. DOI: 10.19586/j.2095-2341.2023.0093
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Streptomyces is widely distributed in various ecological environments as the most advanced actinomycete. It has complex and unique morphological differentiation cycles and strong secondary metabolic abilities. Secondary metabolites of Streptomyces have many biological activities such as anti-infection， anti-tumor， anti-inflammatory and anti-oxidant， immune adjustment and other biological activities， which is one of the main sources of natural active products. In recent years， with the development of marine resources， many new types of marine Streptomyces and their rich secondary metabolites have been discovered. The research on secondary metabolites of marine Streptomyces was reviewed， and the bioactive substances， breeding and fermentation culture of marine Streptomyces were summarized， in order to provide reference and help in shortening the fermentation cycle of marine Streptomyces，improving the yield and activity of secondary metabolites， and developing new marine drugs.

Research and Application Progress in Degradation of Aflatoxin
Yuwei LI, Jiashuo LYU, Yifan YU, Xiaohui LIU, Chengzhen LIU
2023, 13(6): 853-862. DOI: 10.19586/j.2095-2341.2023.0096
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Aflatoxins （AFTs） are potent carcinogens produced by multiple strains of the Aspergillus genus， which show extremely high stability in different harsh environments， so it is also considered to be one of the most important dietary risk factor for humans and animals. Furthermore， other toxic materials may also be generated in the degradation process of AFTs， and the degradation methods might destroy the structure of nutrients， and then decrease the quality of products ultimately. The contamination of aflatoxins places a huge burden on global health systems and the food industry. Although there are many ways to degrade aflatoxins， there is still no way to solve the problem of aflatoxins pollution perfectly， so searching for a technology which can eradicate aflatoxins effectively and safely has become a research hotspot for contemporary researchers. In this article， we summarized the mechanism of aflatoxin toxicity and reviewed several methods of AFTs degradation. We also summarized the advantages and disadvantages of these techniques， in which the research progress of using biological methods and nanomaterials to remove AFTs was recaptitulated systematically. At present， the use of biotechnological means and nanomaterials to degrade aflatoxins has commendable biosafety and efficiency， so the generation of new degradation techniques in the future might revolve around this. Therefore， this paper attempted to support researchers to develop new degradation methods of AFTs.

Application of Rolling Circle Amplification Technique in Electrochemical Biosensors
Yuqi YANG, Xiuxia HE
2023, 13(6): 863-867. DOI: 10.19586/j.2095-2341.2023.0122
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In the design of electrochemical biosensors， signal amplification is an important step in the experimental process， especially when analyzing the sensitivity of the target. Rolling circle amplification （RCA） could obtain a large amount of products in a short period of time and amplify or incubate them on the electrode surface. Through certain design， the electrochemical signal can be quickly amplified. RCA technology has high sensitivity and specificity， while electrochemical biosensors can provide real-time， fast， and low-cost detection. In order to better understand RCA， the basic principle and types of RCA cyclization were introduced， with a focus on summarizing the different technical types and applications of RCA combined with electrochemical biosensors. The development trend of future related research fields was also prospected， aiming to provide reference for the further development and application of RCA technology in electrochemical biosensors.

Molecular Mechanism and Regulation of Pyroptosis
Shuang WEI, Weisong GAO, Jinping DOU, Zepeng ZHAO, Xingjian LIU, Yinyu LI
2023, 13(6): 868-874. DOI: 10.19586/j.2095-2341.2023.0114
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The gene-related pathway of cell death is summarized as programmed cell death. Pyroptosis is a recently discovered mode of programmed cell death that depends on inflammatory caspases and is accompanied by an inflammatory response. The biological characteristics， occurrence and regulation mechanism of pyroptosis are different from other cell death methods. In this paper， we briefly overviewed the research history of pyroptosis， and deliberately introduced the influence factors and regulatory mechanisms of pyroptosis， including non-coding RNA， cell stress， receptor protein and chemical substances， which maybe contribute to clarify the role of pyroptosis in innate immunity.

The Potential and Prospect of Molecular Hydrogen in the Treatment of Skin Diseases
Jianhong YANG, Jun CHEN, Xuefei LI, Lijun LIU, Lili CHEN, Xinsuo DUAN, Shucun QIN, Lei HE
2023, 13(6): 875-881. DOI: 10.19586/j.2095-2341.2023.0087
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The skin， as the largest organ of the human body， plays a crucial role in protecting against physical， chemical， and biological hazards from environment. However， when these hazards exceed the antioxidant capacity of the defense system， it can lead to oxidative stress and chronic inflammation in the body， further damaging collagen fibers and hindering the function of skin cells， resulting in various skin diseases. Molecular hydrogen （H₂） has broad prospects for application in the medical field， including the treatment of skin diseases. This review summarized the antioxidant and anti-inflammatory mechanisms of molecular hydrogen in skin diseases and its administration methods. It also provided an overview of the progress in the application of molecular hydrogen in different types of skin diseases and cosmetic skincare， as well as discussing the safety of molecular hydrogen for the treatment of skin diseases. Molecular hydrogen is expected to be an effective approach for the prevention and treatment of skin-related diseases.

The Role of Mitochondrial Dysfunction in Hypoxic Pulmonary Hypertension
Zhou PAN, Ke HU
2023, 13(6): 882-888. DOI: 10.19586/j.2095-2341.2023.0072
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Mitochondria plays a pivotal role in the pulmonary circulation not only in typical metabolic processes but also in regulating oxidative-reduction signaling， cell cycle progression， cell apoptosis， and mitochondrial quality control， among other noncanonical functions. However， current understanding of these noncanonical mitochondrial functions in the pulmonary circulation and their response to hypoxia is limited. Therefore， there is a need for a deeper understanding of the biological markers and therapeutic targets associated with pulmonary arterial hypertension. This review elucidated the effects of hypoxia on mitochondrial activity and cellular behavior， including the induction of reactive oxygen species （ROS） generation and increased intracellular calcium levels. Furthermore， it highlighted the impact of hypoxia on the expression of pyruvate dehydrogenase kinase and pyruvate kinase M2， as well as the role of mitochondrial fusion and fission in pulmonary artery smooth muscle cell proliferation and resistance to apoptosis. Such investigations contributed to a comprehensive understanding of how hypoxia influences mitochondrial function and how mitochondrial dysfunction affects the progression of PAH. A thorough comprehension of the molecular mechanisms regulating mitochondrial oxidative signaling， metabolism， and dynamic equilibrium holds significant importance in the research and treatment of pulmonary arterial hypertension.

Progress on Treatment of Pulmonary Hypertension Based on Neurohormonal System
Xuesheng FANG, Mingwei BAO
2023, 13(6): 889-894. DOI: 10.19586/j.2095-2341.2023.0092
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Pulmonary hypertension （PH） is a progressive， fatal cardiopulmonary disease， and eventually leads to death due to right heart failure. The pathogenesis of PH has not been clarified and there is a lack of effective treatment for PH. Recent studies have shown that autonomic nervous system （ANS） imbalance and renin-angiotensin-aldosterone system （RAAS） activation are found in PH patients and animal models， which are involved in promoting the occurrence and development of PH and are closely related to the prognosis of PH patients. This paper reviewed the effects and mechanisms of intervention methods targeting the neurohormonal system， including α/β blockers， β3 agonists， pulmonary artery denervation， renal denervation， sympathetic ganglion block， vagus nerve stimulation and RAAS inhibitors on PH， in order to provide a new method for the PH treatment.

Research Progress on RET Oncogene in Thyroid Carcinoma
Jiayuan JIAO, Jiuge XING, Baoshan CHAI
2023, 13(6): 895-899. DOI: 10.19586/j.2095-2341.2023.0002
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Proto-oncogene rearranged during transfection （RET） was identified in 1985， and then， the different types of RET/PTC rearrangement of chromosomes were discovered in papillary thyroid carcinoma （PTC）. Activating point mutations in the RET proto-oncogene were discovered in both hereditary and sporadic forms of medullary thyroid carcinoma （MTC）. Different from the change of RET in PTC， the activation of RET in MTC is mainly through the activation point mutation. Thus， the different types of rearrangement activation of RET are closely related to the high incidence of thyroid cancer. Different activation mechanisms of RET will lead to different treatment strategies. In this review， we discussed the role of RET proto-oncogenes in the pathogenesis， diagnosis and prognosis of PTC and MTC， which was expected to provide reference for the thyroid carcinoma caused by RET activation.

The Research Progress on Action Mechanism of Melanoma Immunotherapy
Pengxiao ZHANG, Nian HU
2023, 13(6): 900-906. DOI: 10.19586/j.2095-2341.2023.0094
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Skin cancer is a common malignant tumor in dermatology in clinical practice， and among which cutaneous malignant melanoma （CM） is the most aggressive one. With the change of people's lifestyle， the incidence of melanoma is gradually increasing， and its development is mainly related to the mutation of melanocytes caused by ultraviolet radiation， which is extremely immunogenicity， and the study of the mechanism of CM development is crucial for the treatment of the disease. This article reviewed the research on immunotherapy of cutaneous malignant melanoma and its related mechanisms of action， with the aim of providing a new way of thinking about the treatment of melanoma.

A Rapid Detection Method Based on qPCR for CRISPR/Cas9 Edited Crops
Xiaotian ZHANG, Zhi WANG, Pengyu ZHU, Shuang WEI, Wei FU, Chunmeng HUANG, Zhihong LI, Huiyu WANG, Yue JIAO
2023, 13(6): 907-912. DOI: 10.19586/j.2095-2341.2023.0080
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Gene editing technology has been widely used in crop breeding and gene function analysis since its birth. CRISPR/Cas9 system has been the most used gene editing technology at present. For detecting the Os11N3 gene of rice edited by the self-developed CRISPR/Cas9 system， this study designed specific Taqman probes for editing sites， combined with rapid genome extraction technology and mature rice PLD gene primer probes. The specificity of the method was verified by simulating different mutations with synthetic plasmids. The system established in this study could save time and cost while obtaining the genome that met the requirements of qPCR detection. It could distinguish at least 1 bp the gene-edited mutant from the wild type， with good specificity and sensitivity. This system can save time and cost for crop breeding and screening， and provide some technical support for gene editing product testing and supervision.

Evaluation of the Anti-disease Effect of Antagonistic Bacterium BJB01 Against Verticillium wilt
Pingcheng DUAN, Kai ZHENG, Yuhong ZHANG, Guoli ZHANG, Guoqing SUN
2023, 13(6): 913-918. DOI: 10.19586/j.2095-2341.2022.0088
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Cotton is an important strategic and livelihood material， which faces many unfavorable factors in the cultivation process， among which cotton Verticillium wilt， as a very harmful plant disease， could cause a large reduction in cotton production. Therefore， a safe and efficient method is needed to deal with the occurrence of cotton Verticillium wilt. We extracted and purified the strain BJB01 from a severely diseased cotton field in Xinjiang. Physiological and biochemical identification and Gram staining test were performed. And we also extracted the DNA of the strain for 16S rDNA sequencing， and blasted the sequencing results on NCBI； then we used the plate standoff test to initially test the effectiveness of the prevention and control； finally， we screened the best prevention and control method and concentration through the greenhouse prevention and control test. Through the test results， the bacterium was found to be 99.71% similar to Bacillus belleiensis， which can be identified as B. belleiensis； and the bacterium showed inhibition of Verticillium dahlia Kleb. in the plate standoff test； through the greenhouse efficacy test， it was found that the bacterium at the concentration of OD₆₀₀=0.6 had the preventive effect of 89.35%， which was better than other methods and treatment concentrations， and the preventive effect of 85.91% was still the best effect in the separate test again. In the comprehensive analysis， Bacillus pelliculatus BJB01 obtained from the cotton disease field in Xinjiang can inhibit Verticillium dahliae， which could be prepared as a biofungicide for cotton production.

ISSR Genetic Diversity Analysis of Bupleurum chinense from Different Populations
Xiaorui YAN, Guozhen XUE, Xiaoxia YANG, Yu WANG, Chenhui DU, Shuosheng ZHANG, Jiquan LIU
2023, 13(6): 919-924. DOI: 10.19586/j.2095-2341.2023.0084
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The genetic diversity of 10 different populations of Bupleurum chinense was studied by inter-simple sequence repeat （ISSR） molecular marker technique， in order to provide basis for further breeding of B. chinense. The DNA of Bupleurum chinense samples from fresh leaves of different populations was extracted by the plant tissue DNA extraction kit. The single factor gradient experiment was used to study the optimal ISSR-PCR reaction system. The DNA amplification products of different populations were subjected to agarose gel electrophoresis， and the genetic diversity of different populations of B. chinense was analyzed by NTSYS-pc software. The results showed that the genetic similarity coefficient of 10 populations ranged from 0.142 8 to 1.000 0. 10 different populations were divided into three categories by cluster analysis， with the first category being Gansu. The second category included five samples of B. chinense， Lingchuan， Henan， Wenxi， Pinglu， and Xiangfen， all belonging to the Jinnan population in Shanxi Province. The third category included four samples， namely Inner Mongolia， Linfen， Hunyuan， and Guangling， belonging to the northern population of Shanxi Province and Inner Mongolia. The 10 populations of B. chinense have relatively abundant genetic diversity and are related to their geographical environment. The results could provide theoretical basis for germplasm resources protection and elite germplasm breeding of B. chinense.

Research and Application of Silver Nanocomposite Film Materials for Cherry Tomato Preservation
Zhaotao ZHANG, Chunhui WANG, Guangye GAO, Hongwei SHI, Xue LI, Guiqiao LIU, Xuefei MAO
2023, 13(6): 925-933. DOI: 10.19586/j.2095-2341.2023.0109
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The application of silver nanoparticles （AgNPs） and other nanoparticles in the preservation of fruits and vegetables has received increasing attention due to their fungicidal and barrier properties.In order to develop efficient green preservation coating films for fruits and vegetables， AgNPs were green synthesized using chrysanthemum extract， and their inhibitory properties against Streptomyces alternifolium were investigated. In addition， composite coating films of AgNPs and chitosan （CTS） were prepared and their effects on the preservation of cherry tomatoes were investigated. It was found that the synthesized AgNPs were spherical， small in size and well dispersed with good inhibitory activity against Streptomyces alternifolium. The freshness preservation test of cherry tomatoes showed that the AgNPs-CTS coated film had better freshness preservation performance than that of the CTS group and the blank group. After 15 days of storage， the AgNPs-CTS coated film had a positive effect on maintaining the high soluble solids， titratable acid and Vc contents in cherry tomatoes， and it could significantly reduce the rotting rate and weight loss rate of cherry tomatoes. The results showed that the nanosilver synthesis method could reduce the use of toxic chemical reagents， fulfill energy saving and environmental protection， and had good bacterial inhibition performance， which could be applied to the postharvest preservation of cherry tomatoes by compositing with chitosan to form a film. The results could provide scientific reference for the development and application of green nanosilver composite freshness coating film.

Preparation of Chondroitin Sulfate and Collagen Peptide of Sturgeon by Multi-enzyme Step-by-step Enzymatic Hydrolysis Under the Condition of pH Gradient
Zhenyu WANG, Wenchao LU, Kangrong ZHONG, Yongjian GUAN, Zhen WANG, Chao CHEN
2023, 13(6): 934-939. DOI: 10.19586/j.2095-2341.2023.0119
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Alkaline protease， papain and bromelain were used to hydrolyze cartilage of sturgeon step by step under pH gradient conditions to prepare sturgeon chondroitin sulfate and collagen peptide simultaneous. The effects of enzymatic hydrolysis temperature， enzymatic hydrolysis time， and enzyme dosage on the efficiency of enzymatic hydrolysis were investigated. Based on comprehensive analysis， the optimal temperature for alkaline protease hydrolysis was 45 ℃， hydrolysis time was 2 hours， and enzyme dosage was 100 U·g^-1 cartilage； the optimal temperature for papain hydrolysis was 50 ℃， hydrolysis time was 2 hours， and enzyme dosage was 50 U·g^-1 cartilage； the optimal temperature for bromelain hydrolysis was 50 ℃， hydrolysis time was 2 hours， and enzyme dosage was 30 U·g^-1 cartilage. Sturgeon chondroitin sulfate was extracted at a rate of 85% with a purity of 93%， while collagen peptide was extracted at a rate of 82% with a purity of 90%. In comparison to the currently used domestic production techniques， the multi-enzyme step-by-step enzymatic hydrolysis process established in this study under pH gradient conditions could improve yield and purity， reduce the amount of alkali， and avoid the introduction of organic solvents， thereby lowering the production cost and lessening the burden of environmental pollution.

Research on Detection Methods of Pseudomonas aeruginosa in Cold Chain Food and Construction of Evolutionary Tree
Zhenhua KE
2023, 13(6): 940-944. DOI: 10.19586/j.2095-2341.2023.0132
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Pseudomonas aeruginosa is a conditionally pathogenic bacterium with greater infection risk and harm to patients. Establishing a detection method for P. aeruginosa in cold chain foods and completing the construction of a traceable molecular evolutionary tree is crucial for food safety. Real-time fluorescence PCR technology was used to establish a rapid detection method for P. aeruginosa in cold chain food. After the detection of P. aeruginosa through cold chain food sampling investigation， the strains detected in the samples were sequenced and analyzed， and a biological evolutionary tree of P. aeruginosa was established to complete the traceability analysis. As a result， a total of 10 pathogenic bacteria were successfully detected from 271 randomly collected cold chain foods using this method， and the overall detection rate of pathogenic bacteria was 3.69% （10/271）. The source of contamination of P. aeruginosa was successfully traced and the localization of various genera was completed. The research results could provide ideas and methods for the detection and traceability analysis of P. aeruginosa and other related pathogens in cold chain food.

Inhibitory Effect of Hydrogen Molecule on Imiquimod-induced Psoriasis-like Dermatitis in Mice
Junkai ZHU, Lingzhi GE, Chao ZHANG, Can CAO, Jiahui WU, Zhen MU
2023, 13(6): 945-953. DOI: 10.19586/j.2095-2341.2023.0112
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Hydrogen molecule is a new type of gas signaling molecule， which has a certain effect on a variety of inflammation diseases. Psoriasis is a chronic， inflammatory disease， and molecular hydrogen is expected to be a new adjuvant therapy for psoriasis. The psoriasis model of mice was induced by 5% imquimod （IMQ） cream for 7 consecutive days， during which the mice were exposed to 35% hydrogen molecular environment twice a day for 2 h each time for intervention. The changes of skin lesions were observed daily and the psoriasis area and severity index （PASI） score was performed. On the 8th day， the skin lesions were taken by death， hematoxylin-eosin （HE） staining， and the histopathological lesion was evaluated using Baker score. The expressions of inflammatory cytokines IL-6， TNF-α and IFN-γ were detected by immunohistochemistry. Real-time quantitative fluorescent PCR （RT-qPCR） and Western blot were used to detect the relative mRNA and protein levels of inflammatory cytokines IL-6， TNF-α and IFN-γ in skin lesions. The content of oxidation index malondialdehyde （MDA） and the activity of antioxidation-related catalase （CAT） were detected by the kit. Results showed that， molecular hydrogen intervention decreased PASI score （P<0.05） and Baker score （P<0.001）， and improved histopathological changes of IMQ-induced mice. The mRNA and protein expression levels of IL-6， TNF-α and IFN-γ in the skin tissue of mice in the hydrogen molecular intervention group were decreased， the content of MDA was decreased， and the activity of CAT was increased （all P<0.05）. The results indicated that， molecular hydrogen can inhibit the formation of psoriasis skin lesions， reduce inflammation and oxidative stress damage， which has the potential to treat psoriasis.

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