Current Biotechnology

Advances on Rhizobium Mutagenesis in Rhizobium-legume Symbiosis System
WANG Yanxia1,2, XIE Zhihong1*
2019, 9(2): 101-107. DOI: 10.19586/j.2095-2341.2019.0002
Abstract ( ) PDF (1965KB)( )

The establishment of rhizobia-legumes symbiotic nitrogen fixation system can not only promote soil nitrogen accumulation and improve soil fertility, but also have great potential for ecological restoration under different environmental factors. However, long-term studies have shown that these environmental factors also limit the system to play a full role in agricultural production. Through mutation breeding, the strain can be genetically mutated to obtain the high-yield and high-quality strains required. Based on this, the research and application of rhizobia mutagenesis breeding with high nitrogen fixation and stress resistance were reviewed in this paper, and the existing problems in the research and application of rhizobia mutagenesis breeding were put forward, expecting to provide reference for nitrogen fixation research.

Progress on the Interaction Between Avr Genes of Pseudomonas syringae and R Genes of Host Plant
HUANG Zhe1, ZHOU Jing2, PAN Sujun1, LIU Jing1, DAI Liangying1, LI Wei1*
2019, 9(2): 108-113. DOI: 10.19586/j.2095-2341.2019.0005
Abstract ( ) PDF (1061KB)( )

Effectors, kind of secreted proteins, are widely existed in bacteria, oomycetes and fungi, which plays an important role in the interaction between pathogenic microorganisms and their hosts. Effectors are classified into avirulence effectors (Avr) and virulence effectors (Vir) according to whether pathogen can compatibly interact with its host or not. This paper introduced the efforts in the typical pathogenic bacteria Pseudomonas syringae and the R genes in host plants, and described the interaction mechanism between Avr genes and R genes, including direct recognition and indirect recognition. Moreover, we discussed and prospected the value of interaction research between Pseudomonas syringae effectors and plant R genes in plant disease resistance breeding.

Genome-wide Identification of Wheat NTL Transcription Factors and Their Expression Patterns Analysis in the Wheat-stripe Rust Interaction
WANG Pei1,2, WANG Fengtao2, CHANG Honglei3, FENG Jing2, GUO Qingyun1*, LIN Ruiming2*
2019, 9(2): 114-121. DOI: 10.19586/j.2095-2341.2019.0004
Abstract ( ) PDF (6170KB)( )

NTL (NAC with transmembrane motif 1-like, NTM1-LIKE) transcription factors belong to the NAC transcription factor family, characterized by a transmembrane domain at the C terminus besides the NAC domain. NTLs have been proved to play important roles in plant development and responding to biotic and abiotic stresses, while the studies of NTLs in wheat are relatively less. Based on this situation, genome-wide identification of NTLs in wheat was carried out, and the phylogeny, gene structure, conserved motifs of NTLs and their expression patterns in the wheat-stripe rust interaction were analyzed. The results showed that there were 17 NTLs in wheat, which belonged to 7 homologous groups, and located on 12 chromosomes. Predictive analysis of subcellular localization indicated that NTLs with localization signals generated in nucleus, plasma membrane, chloroplast thylakoid and endoplasmic reticulum. Besides, some NTLs were induced by the infection of stripe rust in compatible interaction. The results of this study provided a reference for the functional analysis of NTL transcription factor family in wheat.

Detection and Identification of BCMV Infecting Polygonatum kingianum
CHEN Zeli1, YANG Linyi2, CHEN Lu2, SUN Yan2, WEI Zhaoxia2, LI Yongzhong3, ZHAO Mingfu2*, WEN Guosong1*
2019, 9(2): 122-128. DOI: 10.19586/j.2095-2341.2018.0140
Abstract ( ) PDF (10227KB)( )

As an important Chinese medicinal material, Polygonatum kingianum is widely used in many fields. With the increase of its market demand, wild resources of Polygonatum kingianum are already scarce, as a result, the artificial planting industry has been developed. However, the large-area monoculture leads to the serious disease of Polygonatum kingianum. At present, there are many studies on fungal diseases of Polygonatum kingianum, such as leaf spot, anthracnose and black spot, but no report of its viral diseases. In order to ascertain the viral pathogen that infects Polygonatum kingianum and causes the symptoms of mosaic leaf and leaf shrinkage, the diseased samples of Polygonatum kingianum collected from Wenshan in Yunnan province were observed by transmission electron microscope (TEM) and detected with DAS-ELISA and RT-PCR. The leaves of Polygonatum kingianum with obvious infection symptoms were purified to obtain their virus particles, and the shapes of virus were filamentous as observed by a TEM, with the size of (670~760) nm × (9~12) nm. The juice of Polygonatum kingianum samples (DHJ1，DHJ2，DHJ3) with infection symptoms were strongly positive against Bean common mosaic virus (BCMV) antibody detected by DAS-ELISA. Whereafter, total RNA of DHJ-1 which was a positive sample detected by DAS-ELISA was extracted, and amplified by RT-PCR with genera-specific degenerate primers of Potyviridae (Sprimer/M4T), and then the amplified products were sequenced and analyzed. The results showed that the length of obtained sequence was 1 609 bp, including partial nucleosome inclusion body protein b gene (NIb) (588 bp), full-length coat protein (CP) gene (864 bp) and 3′-UTR (157 bp). BCMV belongs to the genus Potyvirus in the family Potyviridae, and the results of homology analysis indicated that the sequence of DHJ1 was 99% and 100% homologous with the nucleotide sequence and CP amino acid sequence of the BCMV Glycine max isolate (KJ807813), respectively, and 97% (at nt level) and 99% (at aa level) homologous with BCMV Arachis hypogaea isolate (HM776124). Phylogenetic analysis based on CP amino acid sequences revealed that the virus DHJ-1 was closely related to the Chinese Glycine max isolate (KJ807813) and Arachis hypogaea isolate (HM776124), and they clustered in one group. It was the first report of virus infecting the genus Polygonatum, which was named as BCMV-DHJ1, indicating that BCMV could naturally infect Polygonatum kingianum.

Review of Biomarkers for Glioblastoma
LIU Mengyu1, XIE Fei1, ZHANG Xin1, ZHAO Pengxiang1,2*
2019, 9(2): 129-138. DOI: 10.19586/j.2095-2341.2018.0118
Abstract ( ) PDF (1154KB)( )

As one of the common primary malignant tumor in central nerve system, glioblastoma(GBM) demonstrates poor prognosis with 14 months of median survival. Higher recurrence is one of the characters of GBM after treatment. The reason for absence of effective therapy of GBM is our poor understanding of tumor biology and mechanism of GBM. As a consequence , it is necessary that comprehensively understanding molecular markers of GBM to improve clinical outcome. This review introduced GBM markers such as EGFR, IDH1/2 and 1p19q as well as imaging biomarkers and immune checkpoints. Accurate classification of GBM and personalized treatment was developed based on these markers. Furthermore, the relevant cancer pathway gives a chance to develop the target treatment and improve the GBM patient survival.

Advances on the Mechanisms of Functional Carbohydrates Against Salmonellosis and Colibacillosis in Livestock and Poultry
QIAO Yu, SHI Bo*
2019, 9(2): 139-145. DOI: 10.19586/j.2095-2341.2018.0109
Abstract ( ) PDF (2267KB)( )

Salmonella enterica (Salmonella spp.) and Escherichia coli are two important zoonotic pathogens, which seriously threaten the health of livestock and poultry, food safety, human health and public safety. With the rapid development of intensive livestock and poultry raising, the harm caused by Salmonella and E. coli is becoming more obvious. Although antibiotics have played important roles in the control of Salmonella and E. coli in livestock and poultry, the abuse of antibiotics leads to drug residues in livestock and poultry products, the emergence of bacterial resistance and direct toxicity to human body. Therefore, it is urgent to use new green feed additives to replace antibiotics in livestock and poultry raising. Researches have shown that oligosaccharides are involved in the occurrence of disease and pathogen infection, and functional carbohydrates have good effects on the prevention and treatment of Salmonella and E. coli. The mechanisms of functional carbohydrates in inhibiting Salmonella and E. coli infections were reviewed in order to provide a reference for the study of functional carbohydrate as antibiotic substitutes used in the prevention and treatment of salmonellosis and colibacillosis in livestock and poultry.

Environmental Fate of Antibiotics and Antibiotic Resistance Genes in Animal Manure
HUANG Rongcai1, GAO Shengtao1, FAN Shijie1, MA Lu1, BU Dengpan1,2,3*
2019, 9(2): 146-151. DOI: 10.19586/j.2095-2341.2018.0137
Abstract ( ) PDF (1063KB)( )

Antibiotics are widely used in animal husbandry due to their outstanding functions, including promoting animal growth, improving feed conversion efficiency, and preventing and treating animal diseases. However, only a small amount of antibiotics can be absorbed and degraded by animals, and most of the ingested veterinary antibiotics are excreted into the environment through feces and urine in the form of prototype or antibacterial metabolites. Apart from antibiotics, manure is also a reservoir of antibiotic resistance genes (ARGs) which contains numerous antibiotic resistant bacteria. Antibiotics and ARGs in the manure can transmit in the environment with the application of manure. Furthermore, residual antibiotics can further enrich ARGs in the environment. It has the potential harm to the human health and ecosystem. Based on this situation, environmental fate of antibiotics and ARGs in animal manure was summarized in order to provide a scientific reference for studying the harm of antibiotics and ARGs to ecosystem and human health.

Preliminary Function Analysis of the MtFVE gene in Medicago truncatula
WANG Ruiliang1, ZHANG Pengcheng2, NIU Lifang2, ZHU Hao3, LI Xuesen3, WANG Xingchun1*, LIN Hao2*
2019, 9(2): 152-160. DOI: 10.19586/j.2095-2341.2019.0014
Abstract ( ) PDF (10563KB)( )

FLOWERING LOCUS VE (FVE), one of the autonomous pathway members in Arabidopsis thaliana, promotes the floral transition by inhibiting transcriptional expression of FLOWERING LOCUS C (FLC) at the chromatin level. FLC acts as a key repressor of oral transition by blocking the expression of integrator genes. Howerver, Medicago truncatula lacks the FLC gene. Therefore, the molecular mechanism underlying the regulation of flowering time by FVE homolog in M. truncatula remains to be elucidated. There were two FVE homologs in Medicago truncatula, named MtFVEa and MtFVEb, respectively, and they both had a typical WD40 protein binding domain. Subsequently, qRT-PCR and semi-quantitative RT-PCR analysis revealed that both MtFVEa and MtFVEb were highly expressed in flower and shoot apices. Yeast double-hybrid assay showed that MtFVEa interacted with MtFLD (FLOWERING LOCUS D, a homologous protein of Arabidopsis FLD) and MtHDA6 (histone deacetylase 6, a homologous protein of Arabidopsis HDA6), suggested that MtFVE may be involved in flowering time regulation by forming the FVE-FLD-HDA6 complex in M. truncatula. The research provided ideas and theory for further research on the function of MtFVE in M. truncatula.

Functional Analysis of Gibberellin Oxidase Gene MtGA20ox in Medicago truncatula
MA Qingxia1§, YIN Pengcheng2§, LIN Hao2, JIN Zhuping1, WANG Huanqing3, YANG Jiangping3, PEI Yanxi1*, NIU Lifang2*
2019, 9(2): 161-168. DOI: 10.19586/j.2095-2341.2019.0009
Abstract ( ) PDF (10129KB)( )

GA20-oxidase (GA20ox) is a key rate-limiting enzyme in the synthesis of gibberellic acid (GA). In order to study the function of GA20ox gene in Medicago truncatula, the amino acid sequence alignment of GA20ox of Arabidopsis thaliana and Medicago truncatula was carried out by bioinformatics methods, and the phylogenetic tree of the families was constructed. The results showed that there were three MtGA20ox, which were MtGA20ox1, MtGA20ox7 and MtGA20ox8, respectively. They had highly homologous to AtGA20ox, and each of them contained a conserved 20G-FeⅡ_Oxy domain. Further, the results of transcript analysis indicated that the expressions of MtGA20ox1, MtGA20ox7 and MtGA20ox8 were tissue specific. Meanwhile, the result of subcellular localization indicated that MtGA20ox1, MtGA20ox7 and MtGA20ox8 proteins were probably localized in nucleus and cytoplasm. In order to comprehend the function of MtGA20ox, two mutants with retrotransposon Tnt1 inserted in MtGA20ox7 and one mutant with retrotransposon Tnt1 inserted in MtGA20ox8 were obtained. The phenotypic observations and data statistics of the three mutants manifested that there were no significant changes in the plant height compared with the wild type. This might be due to the functional redundancy of the three MtGA20ox genes. Results of this study had a certain reference value for studying the function of GA20ox and the regulation of gibberellin on plant height in Medicago truncatula.

Function Analysis of PRP8 Gene of Regulating Cryptochrome CRY2 in Arabidopsis
GUO Yarong1, WANG Yanyan2, LIU Jun2*, PEI Yanxi1*
2019, 9(2): 169-177. DOI: 10.19586/j.2095-2341.2019.0007
Abstract ( ) PDF (9383KB)( )

Plant perceive environment light through photoreceptors to regulate self-growth and development. Cryptochrome 1（CRY1）and Cryptochrome 2（CRY2）are specific blue light photoreceptors in Arabidopsis. CRY2 undergoes blue-light dependent phosphorylation and degradation process, but the mechanism underlie this process is not clear. An early flower mutant was obtained through screening the EMS mutagenesis pools of LUC-CRY2/Col-4 transgene lines. CRY2 protein was over-accumulated in the mutant but the amount of CRY1 protein was not affected. This mutant was named mac1 (more abundant of cry2 1). Map-based cloning and CRISPR/Cas9 knock out experiment showed that this is a new allele of mutation in PRP8, we named it prp8-11. Real-time PCR demonstrated that the accumulation of CRY2 protein is not caused by overexpression of mRNA, but regulated at post transcription level. This study provided new insight for further understanding the function of the PRP8 gene in light signal network.

Promoter Analysis and Realtime-PCR Assay of Denitrifying Genes of Sinorhizobium sp. NP1
ZHANG Yu, WANG Sen, CHEN Duyu, XU Lei*
2019, 9(2): 178-184. DOI: 10.19586/j.2095-2341.2018.0058
Abstract ( ) PDF (2229KB)( )

At present, the release of N2O in sewage denitrification process increases year by year. Lots of N2O cause greenhouse effect, acid rain and destroying the ozone layer. In order to study the mechanism of N2O releasing during denitrification, online bioinformatics software was used to predict the promoter-related information of four denitrifying enzyme genes of Sinorhizobium sp. NP1. It has been predicted that the gene expression of the four key enzymes required for the denitrification process do not use the same promoter, but four different promoters. Meanwhile，the promoters of nitrate reductase and nitrite reductase are strong, while promoters of nitric oxide reductase and nitrous oxide reductase are weak. In order to explore the above prediction results, we used real-time PCR to detect the difference in transcription levels of four denitrifying genes in Sinorhizobium sp. NP1 cultured with KNO3 as the sole nitrogen source. And we measured the content of metabolites produced during denitrification. It was found that the transcription levels of nitrate reductase gene and nitrite reductase gene were significantly higher than those of nitric oxide reductase gene and nitrous oxide reductase gene. And there was a lot of N2O in the denitrification process. We speculated that it may be related to the strength of different promoters. Therefore, the expression of denitrifying enzymes can be regulated by genetic engineering such as transformation of operons, which lead all N2O reduced to N2 and then released into the air. In a word, building more efficient and less polluted denitrifying bacteria has profound social ecological significance.

Identification and Analysis of a Cellulase Hyperproducing Mutant of Humicola insolens
CHEN Yuan1,2, LI Jinyang2, ZHANG Yuhong2, LIU Bo2, ZHANG Zhiwei1*, XU Xinxin2*, ZHANG Wei2
2019, 9(2): 185-190. DOI: 10.19586/j.2095-2341.2019.0012
Abstract ( ) PDF (2220KB)( )

Humicola insolens holds a high capacity for neutral and thermostable cellulases secretion that has industrial applications promising, like biomass energy, feed and textile industry. In this study, we isolated a mutant T53 with enhanced cellulase and hemicellulase activities from a transfer DNA (T-DNA)-tagged mutant library of H. insolens that was developed before. The FPase, endoglucanase, cellobiohydrolase, β-glucosidase, and xylanase activities of T53, measured at the end of fermentation, were 40%, 60%, 90%, 10%, 60% higher than those of the wild-type strain, respectively. The result of SDS-PAGE showed that the secretion of extracellular proteins, especially cellulases and hemicellulases of T53 strain was significantly higher than that of wild type strain. The sequence flanking the T-DNA insertions in T53 was isolated and a putative zinc finger protein encoding gene potentially involved in cellulase and hemicellulase expression was identified. This study laid an important foundation for revealing the molecular mechanism of cellulases high-yielding in mutant T53, which would also enrich our understanding of the genetics background of H. insolens.

Degradation of Patulin in Grape Juice by Glow Discharge Plasma
MA Yayun1, LONG Haitao1, DU Mingyuan1, XUE Huali1, NAN Mina1, FU Guorui1, BI Yang2, PU Lumei1*
2019, 9(2): 191-199. DOI: 10.19586/j.2095-2341.2018.0106
Abstract ( ) PDF (1668KB)( )

In order to improve the security quality of grape juice, the grape juice containing patulin (PAT) was treated with a glow discharge plasma (GDP) reactor, and the optimal process conditions were determined by single factor experiment and orthogonal experiment: The voltage was 560 V, the treatment time was 3 min, the concentration of PAT was 3 mg/L, and 100 mg/L H2O2 was used as the catalyst. Under these conditions, the degradation rate of PAT in grape juice by GDP was as high as 98.9%. The results of degradation kinetics indicated that the degradation of PAT in grape juice by GDP conformed to the first-order kinetics. In addition, the physicochemical indexes of grape juice were evaluated after GDP treatment. The results showed that the reducing sugar, total soluble solid (TSS), total acid, pH, total phenols and total flavonoids of grape juice treated with GDP did not change significantly, and the clarity of grape juice was improved to a certain extent. Results of the research provided a theoretical basis for the application of glow discharge plasma to the degradation of patulin in grape juice.

Study on Isolation and Fermentation Performance of Dominant Lactic Acid Bacteria During the Processing of Longxi Bacon
DENG Zhanrui, YUN Jianmin*, GUO Juan, NIU Yaoxing, LI Yanhu
2019, 9(2): 200-209. DOI: 10.19586/j.2095-2341.2018.0098
Abstract ( ) PDF (3219KB)( )

Longxi bacon is a traditional fermented meat products. Using Longxi bacon as the raw material, the dominant lactic acid bacteria (LAB) during the processing were isolated and identified by the methods of classic morphological classification, physiological and biochemical identification. Then the strains with excellent fermentation performance were screened by a series of tests, including acid-producing ability, growth characteristics, tolerance to sodium chloride and sodium nitrite, capability of producing gas, mucus, protease and lipase. Whereafter, raw meat was fermented by artificially inoculating the superior strain in order to analyze its effect on the sensory quality of bacon. The results showed that seven strains of LAB screened from Longxi bacon were identified as Lactobacillus plantarum, L. sake, Lactococcus lactis, Pediococcus pentosaceus, Leuconostoc mesenteroides, L. alimentarius and L. curvatus, respectively. Among them, the strain R-1 which was identified as Lactobacillus plantarum had strong acid-producing ability, fast growth rate and good tolerance to sodium chloride and sodium nitrite. The simulating fermentation test of bacon revealed that the flavor of Longxi bacon had been improved after inoculating strain R-1, and it showed better sensory acceptance. The research indicated that Lactobacillus plantarum R-1 had good fermentation performance，and it could be used as a source of strain for the modernization of traditional fermented bacon.

Optimization of Liquid Fermentation Conditions for the Production of Nattokinase from Soybean Milk
NI Nan1,2, XIN Zhihong1, ZHANG Lijing2, WANG Fengzhong2*, LI Shuying2*
2019, 9(2): 210-215. DOI: 10.19586/j.2095-2341.2018.0138
Abstract ( ) PDF (1178KB)( )

Nattokinase is an ideal natural drug for the prevention and treatment of thrombosis. Liquid fermentation can improve the yield of nattokinase. Taking Bacillus subtilis natto as the start culture, soybean milk as the fermentation medium, nattokinase activity as function evaluation index, the slag rate and finial pH as auxiliary indexes, the effects of fermentation time, liquid loading, bacteria loading and initial pH on nattokinase activity were studied by single factor and orthogonal analysis. The optimal fermentation conditions were determined as follows: The fermentation time was 30 h, the liquid volume in flask was 5%, the amount of bacteria was 1.75%, and the initial pH was 5.5. The nattokinase activity was 429 869.34 IU/mL under this condition, which was 5.41 times of that before optimization. It can be seen that the orthogonal test successfully optimized the liquid fermentation process of Bacillus subtilis natto and significantly increased the expression of nattokinase, which laied a theoretical foundation for the development of functional food.

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