Biotechnology Bulletin

Research Progress on Plant miR390

XIE Jie ,WANG Ming ,LI Qing ,PAN Fei ,XIONG Xing-yao ,QIN Yu-zhi

2018, 34(6): 1-10. doi:10.13560/j.cnki.biotech.bull.1985.2017-1093

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MicroRNAs（miRNAs），a group of highly conserved non-coding RNAs in eukaryotes，regulate target gene expression via multiple approaches of slicing target mRNA and inhibiting translation of target mRNA，i.e.，regulations of target genes are achieved at post-transcriptional level. Therefore，they play important roles in plant organ morphogenesis，growth and development，signal transduction and abiotic stress response. One of the vital miRNA family is miR390，which is an ancient and highly conserved miRNA family；its target gene，AGO7，is the basic component of RNA-induced silencing complex（RISC）that extensively is involved in cutting target mRNAs，and plays important role in plant growth，lateral organ polarity morphogenesis，floral organ morphogenesis，and stress response. The previous studies on miR390 mostly were focused on plant growth and development，while the function to abiotic stress has been little reported. This review summarizes the discovery and the types of miR390，the formation process of miR390 family，the role of miR390 family in plant growth and development，and the plant responses to abiotic stresses，such as heavy metals，drought，salt，low temperature stresses，etc.. Meanwhile，the article prospects the approaches for studying miRNAs，which is useful for comprehensively understanding the research progress of miR390 and its function in abiotic stresses in plants.

Research Progress on Whole-genome Sequencing on Important Domesticated Animals

LI Xiao-kai ,WANG Gui ,QIAO Xian ,FAN Yi-xing ,ZHANG Lei ,MA Yu-hao ,NIE Rui-xue ,WANG Rui-jun ,HE Li-bing ,SU Rui

2018, 34(6): 11-21. doi:10.13560/j.cnki.biotech.bull.1985.2017-0719

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Whole-genome sequencing mainly refers to obtain the full genome sequence map of a species through different sequencing and assembly alignment method，and based on it to explore their genetic diversity，selection signal，and the origin and evolution of representative individuals or groups by constructing whole-genome genetic variation map. Using single nucleotide polymorphism（SNP），insertion and deletion（Indel）and copy number variation（CNV）genetic variation as molecular markers，the studies on whole-genome sequencing has been achieved a lot in the origin and evolution of domestic animal，domestication，adaptive mechanism，candidate genes for important economic traits，and population demographic. This paper focuses on the key achievements of whole-genome sequencing on common domesticated animals（such as pig，horse，cattle，sheep，goat，and closely-related species），and discusses the advantages，disadvantages and significance of whole-genome sequencing in production. In addition，this paper summarizes and prospects the future development of researches on whole-genome sequencing. And we also hope to provide a reference for mapping functional genes with important economic traits and the origin domestication of domesticated animals.

Research Advances on the Physiological Functions of Follistatin

LI Hao-yu ,HE Xiao-yun

2018, 34(6): 22-29. doi:10.13560/j.cnki.biotech.bull.1985.2017-0991

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Since the discovery of follistatin（FST）in follicular fluid of pigs and cattle in the 1980s，studies on FST have never ceased and various functionalities of FST have been identified by in vivo and in vitro experiments. FST as a secretory protein is present and secreted in various tissues and organs of mammals. It plays a key role in hormone regulation，energy metabolism and muscle adipose tissue proliferation and differentiation. As FST is found from the gonadal，people have clearly studied FST regulating sex hormone secretion and signal transmission and promoting oocyte maturation and embryonic development，and the animal experiments with a number of species were carried out to identify the mechanism of action. FST as an inhibitor of TGF-β superfamily can be specifically combined with a member of myosin（MST），which produces antagonism and promotes muscle proliferation，differentiation and expansion. Brown fat as an important metabolic tissue in mammals has been a hot spot. FST has been found to promote brown adipose differentiation and induce white fat browning in recent years，which has been validated in cell experiments and mouse experiments. This review introduces FST and its important functions as well as the action mechanisms and development prospects of varied function，aiming at paving the basis for further research and development of FST.

Research Advance on Oral Vaccine for Aquatic Animals

LIU Shi-xu ,WANG Qing ,FANG Zhen-zhen ,CHANG Ou-qin ,ZENG Wei-wei ,HUANG Zhi-bin

2018, 34(6): 30-37. doi:10.13560/j.cnki.biotech.bull.1985.2017-0805

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The application of vaccines may effectively improve their resistances against various common diseases in aquaculture，thus decrease the possibility of the incidence of diseases and infections，reduce the use of chemical drugs，eliminate the environmental pollution，and even decrease economic losses from environmental governances. Vaccination by injection is time-consuming and easily causes injuries to fishes. Vaccination by immersion needs high dosages and thus easily results in strong stress reaction. The oral vaccine can be applied to any variety and size of fish，thus may avoid the problems by injection or immersion，and it is an ideal immune approach. Among the present oral vaccines，the adjuvants like polymeric microspheres and biofilm used to prevent the antigens in vaccine from the effects of all digestive enzymes are widely applied；however，the cost is quite high in field application. Biological carrier can be a genetic engineering vaccine with antigens delivery work by non-pathogenic microorganisms，such as bacteria. These biological carrier vaccines are actually in low costs for application and induce a solid immunization effect，however，the cycle of safety evaluation is long and there is difficulty in large-scale production. This paper reviews the status and key technologies of different forms and formulations of main oral vaccines. Also the paper prospects that the study on aquatic animal oral vaccines should focus on vaccine safety and applicability in the future research work，and developing oral vaccine simultaneously in lower cost and better immunization effect.

Application of Selection and Optimization of Promoter in Metabolic Engineering of Saccharomyces cerevisiae

WANG Ke-wen ,YIN Xue, WANG Yu ,LI Yu-hua

2018, 34(6): 38-47. doi:10.13560/j.cnki.biotech.bull.1985.2017-0909

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Saccharomyces cerevisiae，the simplest eukaryote，is widely used in the life science as a model organism. So far，the majority of the natural products were extracted directly from organism，which has been gradually replaced by the approaches of synthetic biology duo to its inefficiency and consuming a lot of biological resources. Modifying its metabolic pathways and adding heterometabolic pathways of S. cerevisiae for target natural products has been an efficient approach of acquiring resources. The direct synthesis of target metabolites has become the research hot spot in synthetic biology and metabolic engineering of S. cerevisiae through optimizing and transforming the exogenous gene promoter，regulating the expression level of the exogenous gene in the host，and coordinating the metabolic pathways of hosts. Here we had a detailed introduction on the structures and types of promoters as well optimizing methods of expression in the process of synthetizing natural products from the constructed S. cerevisiae，which may provide a reference for relevant researchers to use S. cerevisiae as chassis cells in synthetic biology.

SMRT Sequencing and Its Application in Microorganism Studies

TANG Yong ,LIU Xu

2018, 34(6): 48-53. doi:10.13560/j.cnki.biotech.bull.1985.2017-1009

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The development of high-throughput sequencing has provided power for researchers to further explore the microorganism. Along with rapid advances of third generation sequencing（TGS）technologies，especially single molecule real time sequencing（SMRT）developed by Pacific BioSciences（PacBio），methods of studying micro-organism are going through a revolutionary change. SMRT sequencing technology，for its special SMRTbell sequencing libraries and super-long reads length，provides a new choice for sequencing full length of 16S rRNA，and also provides a novel method for acquiring accurate and reliable metagenome and whole genome sequence of microbial species. With the cost of sequencing using SMRT sequencing technology remarkably reduced，a growing number of microbial studies will be performed with the PacBio series platform based on SMRT technology，including sequencing of 16S rRNA gene，metagenome sequencing，and whole genome sequencing. Here，we reviewed the principle and characteristics of SMRT sequencing technology and its application in full length sequencing of 16S RNA，metagenome sequencing，and transcriptome sequencing of microorganism. Further we analyzed the advantages and issues while SMRT sequencing technology is used in studying microorganism，as well as the potential issues of download analysis of SMRT reads. This review is aimed to provide advice or reference for researchers who will employ SMRT sequencing technology into microbial studies.

Studies on Total RNA Extraction Methods from the Stems of Dendrobium Species

SHAN Ting-ting ,CHEN Xiao-mei ,GUO Shun-xing ,WANG Qian-qing ,WANG Ai-rong

2018, 34(6): 54-58. doi:10.13560/j.cnki.biotech.bull.1985.2017-0920

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This work is aimed to establish a fast and efficient method of extracting total RNA from the stem of Dendrobium species for further molecular biology investigation. Seven methods were used to extract the total RNA from the stem of Dendrobium officinale，then gel electrophoresis and UV spectrometer were applied to determine the quality of the extracted total RNA，and the screened method was verified by samples of D. officinale of different growth conditions and times，and of 5 species of Dendrobium. Results showed that the integrity，purity and extraction capacity of the stem total RNA of D. officinale by Improved HUAYUEYANG Quick RNA Isolation Kit（（M7）was the best. The stem total RNA，extracted using M7，of D. officinale，of D. nobile，of D. chrysotoxum，of D. thyrsiflorum and of D. hercoglossum，and of D. officinale planting in plastic house and greenhouse for 1 year respectively，as well as in plastic house for 3，12 and 24 months respectively，were verified to meet the quality requirements on the integrity，purity and extraction capacity. In conclusion，M7 is suitable for stem total RNA extraction of Dendrobium species for easy handle and fine repeatability.

Detection of Escherichia coli O157 by Reverse Transcriptase Loop-Mediated Isothermal Amplification

LIANG Yu-lin, LIU Xiu ,ZHOU Peng-fei ,ZHOU Zhen-sen, YIN Jian-jun

2018, 34(6): 59-65. doi:10.13560/j.cnki.biotech.bull.1985.2017-0932

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A reverse transcriptase loop-mediated isothermal amplification（RT-LAMP）method was developed to specifically detect Escherichia coli O157. A number of primers were designed for specific conservative rfbE gene in E. coli O157. A real-time fluorescent RT-LAMP for detecting E. coli O157 was established by optimizing primer selection and reaction conditions. The specificity and sensitivity of the method were verified by using E. coli O157 and its artificial contaminated skim milk samples，and compared with the sensitivity of rRT-PCR（real-time fluorescent quantitative reverse transcription polymerase chain）. The RT-LAMP reaction was completed within 30 min under the condition of isothermal temperature of 65℃. RT-LAMP was highly specific since there was no specific amplification except two strains of E. coli O157. In the detection of artificial contaminated skim milk samples，the sensitivity reached 20 CFU/g，10 times of that by the rRT-PCR，meaning this method was highly sensitive. The established real-time fluorescent RT-LAMP will provide a powerful means for rapid detection of E. coli O157 on site.

cDNA Cloning and Transcriptional Expression Analysis of OsMPK15 in Rice

SHI Jia YANG Dan-dan GE Hui-wen DU Jing-yao LIANG Wei-hong

2018, 34(6): 66-72. doi:10.13560/j.cnki.biotech.bull.1985.2017-0978

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MAPK gene involves in the rice’s responses to biotic and abiotic stress as well as its development and growth. Cloning rice OsMPK15 and preliminarily studying its expression would provide foundation for further its functional studies. In this study，RT-PCR was used to clone its cDNA coding region of OsMPK15 from rice Nipponbare roots，and quantitative real time PCR（qRT-PCR）to analyze the expression characteristics in various tissues and under different abiotic treatments. The results showed that the cDNA coding sequence of OsMPK15 isolated from rice Nipponbare root was identical with the results of bioinformatics prediction. Sequence analysis revealed that a highly conserved serine/threonine kinase domain in MAPK protein was located at the position from 13th to 304th in OsMPK15 amino acids sequence，and it belonged to group E of MAPK family. qRT-PCR results demonstrated that OsMPK15 gene was widely expressed in various tissues，especially accumulated in leaves and leaf sheaths at young panicle stage. The expression of OsMPK15 gene in seedling roots was up-regulated by drought and salt stress with different expression pattern. Meanwhile，the treatment with stress-related hormones ABA，JA and SA resulted in the responses and the highest up-regulation by ABA.

Identification and Pathogenicity Analysis of Cotton Verticillium Wilt from Shihezi Region of Xinjiang

ZHANG Meng-tian, PEI Juan, LI Guo ,ZHAO Hui ,CHEN Jian-quan, ZHU Jian-bo ,WANG Ai-ying

2018, 34(6): 73-78. doi:10.13560/j.cnki.biotech.bull.1985.2017-1043

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This study is aimed to investigate the genetic variation and pathogenic differentiation of cotton verticillium wilt in Shihezi region of Xinjiang. By the microscopic observation of isolates，the cloning of ITS sequences and the construction of molecular phylogenetic tree，the isolation and identification as well as pathogenicity of various cotton verticillium wilt from different hosts were analyzed. The results showed that the isolated 24 strains of cotton verticillium wilt all belonged to Verticillium dahliae，of which 11 strains were deciduous cotton verticillium wilt and the rest were non-deciduous cotton verticillium wilt. The results of molecular phylogenetic tree showed that 24 strains belonged to two different branches. The infection results to the same host showed that the pathogenicity of 24 strains was classified into three types：strong，moderate，and weak. The strong one was 75%，and it was 23.3% higher than that of 51.7% in 2010，indicating that the pathogenicity types of cotton verticillium wilt in Shihezi region changed greatly and more virulent strains appeared；this probably was correlated with many years planting cotton in Shihezi region. Among them，SHZ-9 was the most virulent while SHZ-4 was the weakest pathogenic strain，indicating that there was obvious pathogenic differentiation of cotton verticillium wilt in Shihezi region. In conclusion，the different virulence strains could be divided into three nutrient affinity groups（VCGs）. The results of this study demonstrated that the pathogen of cotton verticillium wilt in Shihezi region of Xinjiang was Verticillium dahliae，the strong virulence strains increased and the genetic differentiation of strains was significantly different.

Preparation of Polyclonal Antibody Against Ratoon Stunting Disease and Its Immunomagnetic Beads

GUO Ying ,WANG Wen-hua ,LIU Li-qing ,HU Min

2018, 34(6): 79-83. doi:10.13560/j.cnki.biotech.bull.1985.2017-1012

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This study is to isolate sugarcane Lxx by preparing Lxx immunomagnetic beads（IMB）for the further study the interaction between pathogen and sugarcane. The pathogen of Lxx was isolated from the RSD PCR-verified infected sugarcane for the preparation of polyclonal antibody. Then the Lxx polyclonal antibody was coupled with magnetic Fe3O4 nano particle for preparing IMB. The isolates were identified as Lxx according to the morphological characteristics of colony and the PCR results. The fine polyclonal antibody was obtained by immunizing rabbits with Lxx. The titer of the polyclonal antibody against Lxx was greater than 204，800 by indirect ELISA. The appearance of the IMB was globular，and the particulates were 125-268 nm in diameter. The isolates with high affinity for Lxx in the culture media can be used for PCR reaction. The successful preparation of Lxx IMB can rapidly isolate and enrich Lxx，which provides a convenient and efficient technique for the detection of pathogenic bacteria under the interaction of Lxx and sugarcane.

Cloning of a HD-ZIP Transcription Factor Gene HD20 and A Preliminary Study on Function of Fiber Differentiation in Tobacco

GAO Jie ,TONG Shuo-qiu ,ZHONG Jie ,GONG Xian, WU Yong-jun

2018, 34(6): 84-89. doi:10.13560/j.cnki.biotech.bull.1985.2017-0928

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To understand the role of HD20 in the specification of fiber differentiation，we cloned the HD20 gene from tobacco by RT-PCR method. The cDNA sequence was cloned according to specific primers designed by the genomic database in tobacco and named asHD20（GenBank accession number：KC339003）. Sequence analysis showed that the cDNA sequence of HD20 was 762 bp and encoded 253 amino acids. Subcellular localization assays showed that it appeared in the nuclei. The HD-ZIP protein contained two significant domains，including the HD domain with 60 amino acids at the N-terminus and a ZIP matrix with 28 amino acids. We introduced HD20 into the expression vector pSH-737，transferred it by Agrobacterium mediated-dip flower method，and obtained positive transgenic plants for paraffin sections. The results of paraffin section showed that the xylem of stem was more developed than the control group. In conclusion，the result provided evidence that HD20 plays a role in the spatial control of fiber differentiation. The analysis of the regulatory effect of HD20 on tobacco and Arabidopsis helps to create a theoretical base for studying HD20 function.

Effects of Leaf Spraying Fertilizer on Mineral Elements in Wheat Grain

LI Wen-zong, LI You-fang ,Li Wei-hua ,WANG Lei

2018, 34(6): 90-95. doi:10.13560/j.cnki.biotech.bull.1985.2017-1079

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Iron（Fe），zinc（Zn）and selenium（Se）are essential trace mineral elements in the organism and play an important role in the growth and development of plants，animals and human body. This work is to increase the contents of Fe，Zn and Se in wheat grain and further analyze the effects of iron，zinc，selenium fertilizer and their combinations on the contents of iron，zinc，selenium and other mineral elements in wheat grain，meanwhile to explore the most appropriate combination of foliar spraying. We used different concentration of Fe（ferrous sulfate），Zn（zinc sulfate）and Se（sodium selenite）fertilizers and their combinations to be sprayed to the leaves of wheat cultivar BJ0045. The results showed that spraying Fe，Zn and Se fertilizers significantly increased the contents of Fe，Zn and Se in BJ0045 grain. Fe and Zn promoted each other’s absorption. Zn enhanced the absorption of Se and Fe，whereas Se inhibited Zn absorption slightly. The mixed Fe/Zn/Se fertilizer significantly raised the content of Fe，Zn and Se in wheat grain among all combinations. we also found that the different combinations of foliar spray affected differently the absorptions of Ca，Mg，Cu，Mn，and P mineral elements in the grain.

Photosynthesis and Chlorophyll Fluorescence Characteristics of Ophiopogon japonicus（L.F.）Ker-Gawl Under Different Shade Conditions

LIU Li-juan ,GAO Hui

2018, 34(6): 96-101. doi:10.13560/j.cnki.biotech.bull.1985.2017-0894

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Different plants have varied physiological characteristics，thus their irritability to light is also different. Therefore，plants form unique light requirements to adapt themselves to their own growth during evolution. Photosynthetic index and leaf green fluorescence parameters of Ophiopogon japonicus（L.F.）Ker-Gawl leaves were measured by LI-6400XT portable photosynthesis analyzer under 5 light intensity treatments（light transmittance of 95%，72%，48%，24%，and 8%）. With the increase of relative light intensity，the apparent quantum yield（AQE）presented the trend of first increase and then decrease with the increase of light intensity. O. japonicus（L.F.）Ker-Gawl leaf pigment content and initial fluorescence intensity（F0）increased. Chlorophyll ab value was also in the trend of first increase and then decrease. Fluorescence parameter PSⅡ maximum light energy conversion efficiency increased. The actual PSⅡ photochemical efficiency（ΦPSⅡ）under the action of light，PSⅡ effective photochemical quantum efficiency（Fv’/Fm’），electron transfer rate（ETR），and pho-tochemical quenching coefficient（qP）% had a rising trend under the relative light intensity of 72%-48%. Under the relative light intensity of 72%-48%，the response of O. japonicus（L.F.）Ker-Gawl to PSⅡ was stronger，which was conducive to the growth and development of O. japonicus（L.F.）Ker-Gawl. Therefore，appropriate shade measures should be employed in large-scale cultivation of O. japonicus（L.F.）Ker-Gawl.

Analysis of Effects of Body Sizes on the Weight of Polled Yak

PEI Jie ,WANG Hong-bo ,CHU Min ,ZHA Xi-zhuo-ma ,BAO Peng-jia ,LIANG Chun-nian ,LUO Zheng-jie ,WU Pu-de ,DING Xue-zhi ,WU Xiao-yun ,YAN Ping ,GUO Xian

2018, 34(6): 102-108. doi:10.13560/j.cnki.biotech.bull.1985.2017-0806

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The aim of this study is to accurately estimate the weight of polled yak by its body size，and to subdivide the effects of body parts’ sizes on the weight. In the experiment，247 polled yaks aging 8 to 10 months，155 male yaks and 92 female yaks，were randomly selected as objects of the study. Four body size data of body length，standing height，chest circumference，and cannon bone circumference，as well as a yak’s weight were measured. Multiple linear regression equations were established by stepwise multiple linear regression. Then the direct and indirect effects of each body size on weight were calculated by path analysis. The results suggested that the correlation between chest circumference and cannon circumference of male yak did not reach significant difference（P >0.05），and the phenotypic correlations among other traits reached extremely significant difference（P < 0.01）. There was no significant difference between the estimated values and the observed values of the weight. The direct effect of chest circumference on the weight was greater than the effects of the other body parts’ sizes，and greater than the indirect effect of chest circumference through the other body parts’ sizes. In conclusion，the multiple linear regression equations can be used for selective breeding practice in polled yak，and the path analysis suggests that the dominant factor of impacting polled yak weight was the chest circumference.

Cloning of Carboxylesterase Gene cDNA Fragments in Sitobion avenae（Fabricius）and Its Expression Analysis Under Imidacloprid Stress

BAI Wei-wei ,GAO Hai-feng, ZHANG Hang ,YANG An-pei ,LI Guang-kuo

2018, 34(6): 109-114. doi:10.13560/j.cnki.biotech.bull.1985.2018-0169

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Carboxylesterase plays an important role in insect detoxification and metabolism processes of insecticides. This research aims at analyzing the expression levels of carboxylesterase gene in Sitobion avenae（Fabricius）under imidacloprid stress. Homologous cloning strategy was used to clone carboxylesterase gene cDNA fragment，and its expression variations under different dose of imidacloprid were analyzed with qRT-PCR. A carboxylesterase gene cDNA fragment of 392 bp，named SaEST 3（GenBank accession number is KY 441614），was amplified from S. avenae（Fabricius）using homologous cloning strategy. The gene sequence coded a polypeptide of 130 amino acid residues，the calculated molecular mass was 14 kD and the theoretical isoelectric point（pI）was 4.93. Homology alignment and bioinformatics analysis suggested that the amino acid sequence of SaEST3 was highly identical to that of carboxylesterase gene from Acyrthosiphon pisum（94%），Diuraphis noxia（85%），Aphis nerii（80%），and Myzus persicae（80%）. The results of qRT-PCR showed that the relative expression of SaEST 3 mRNA was up-regulated under different dose of imidacloprid. In a summary，the cloned sequence was carboxylesterase gene of S. avenae（Fabricius），and imidacloprid stress may affect the expression of SaEST 3 gene in S. avenae（Fabricius）.

Activity Variations of Protective Enzymes in Paracoccus marginatus After Fed Different Cassava Cultivars

WANG Ya-ru ,LIANG Xiao ,WU Chun-ling ,CHEN Qing ,ZHAO Hui-ping

2018, 34(6): 115-119. doi:10.13560/j.cnki.biotech.bull.1985.2018-0054

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To explore the mechanism of Paracoccus marginatus selecting host plants，the activity variations of peroxidase（POD），polyphenol oxidase（PPO），catalase（CAT）and superoxide dismutase（SOD）in P. marginatus after fed different cassava cultivars for 48 h were determined by ultraviolet spectrophotometry. The results showed that the enzymes’ activities in adult females fed with cultivar C1115，Swiss F21 and Myanmar were significantly lower than those fed with the control cultivar BRA900（P ﹤0.05），the decreasing range were listed as：41%-43%（POD），60%-63%（PPO），35%-55%（SOD）and 54%-60%（CAT），while those in adult females fed with cultivar SC205 and ZM9066 did not show significant difference. Above results indicate that cassava cultivar C1115，F21 and Myanmar show strong inhibitory effects on the activities of POD，PPO，SOD and CAT in P. marginatus，while BRA900，SC205 and ZM9066 do not show significant inhibitory effects on those protective enzymes，which inclines them to be damaged by P. marginatus.

Properties of Extracellular Protease of Microbe DH-2 from Mangrove and Optimization of Enzyme Producing Conditions

XU Shan ,LI Ren-qiang ,ZHENG Zhen-hua ,ZHANG Yun ,SUN Ai-jun ,HU Yun-feng

2018, 34(6): 120-127. doi:10.13560/j.cnki.biotech.bull.1985.2017-1034

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This work is aimed to isolate the strains producing proteases from Daya Bay mangrove and to characterize the enzymatic properties and the optimal fermentation conditions. The clear zone method was used to screen strains，the Folin-phenol reagent method to determine the activity of protease，and the single factor experiments and the orthogonal experiments to determine the optimal fermentation medium and the fermentation conditions. Protease-producing Bacillus subtilis DH-2 was isolated from soil samples，and the optimal fermentation pH and temperature of the protease secreted by DH-2 were 8.0 and 65℃，respectively. The protease remained over 80% of its original activity after treatment for 60 min at 50℃. The protease tolerated multiple metal ions，organic solvents and surfactants. The optimal fermentation conditions for the protease were：1%（m/V）soluble starch，1% tryptone，1% NaCl，the initial pH of 5.5，and the inoculation amount of 7%，36 h culture at 40℃. Under optimal fermentation conditions，the enzyme activity of the fermented liquid was 236.30 U/mL，which was about eight times of the activity in initial screening. Conclusively，the protease has a relatively wide range of action temperature and pH，and exhibits fine resistance to multiple metal ions，organic solvents and surfactants，and is characterized stably.

Isolation and Screening of Anti-MRSA Actinomycetes in the Soil from the Primitive Tropical Rainforest Yingge Ridge

PAN Jie-ming ,ZHANG Rong-yi ,DENG Jia-ai ,TAN Zhi-qiong

2018, 34(6): 128-133. doi:10.13560/j.cnki.biotech.bull.1985.2017-1085

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Yingge Ridge is a primitive tropical rainforest located in Ledong County，Hainan Province. In this study，the actinomycetes were isolated from the Yingge Ridge soil and screened for antibacterial activities against methicillin-resistant Staphylococcus aureus（MRSA）. Actinomycete strains were isolated by plate dilution，and MRSA was used as the target to test antibacterial activities. Strains with antibacterial activity were subjected to 16S rDNA sequence determination. The antibacterial activity of crude extract of strain was determined by filter paper method，and the active substance of crude extract was detected by high performance liquid chromatography. A total of 168 strains of actinomycetes were obtained from the soil of the Yingge Ridge. Ten of them had strong anti-MRSA activity，and the inhibition zone of the crude extract of the fermentation broth was between 8 mm and 25 mm. The identity of 16S rDNA sequences from 10 strains of anti-MRSA Actinobacteria was over 99% in comparison with those of Streptomyces in Genbank. By HPLC fingerprints of 10 strains of fermentation broth，the secondary metabolites of strain 7，5，and 9-1 were abundant，and the activity test showed that the growth of MRSA was obviously inhibited，which provided a certain scientific basis for further research.

Effects of Different Signal Peptides and Their Molecular Chaperones on the Secretion of Neutral Protease in Bacillus subtilis

YANG He-bao ,HU Mei-rong ,ZHENG Xiang ,MOU Qing-xuan ,GAO Pei-ru

2018, 34(6): 134-140. doi:10.13560/j.cnki.biotech.bull.1985.2017-0989

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By screening five different signal peptides to replace the original signal peptide of the plasmid pHT43-npr and integrating two different molecular chaperones（PrsA and DnaK）to the plasmid pHT43-npr，we constructed recombinant plasmids and then transformed them to Bacillus subtilis WB800N to have induced expressions. As a result，five recombinant strains with new signal peptide were constructed，no hydrolyzed circles emerged via milk plate testing，and all the five strains’ enzyme activities were obviously lower than that by control strain. We also successfully constructed two recombinant strains integrated the molecular chaperones，both of the two strains emerged hydrolyzed circles in milk plate testing. Both strains had neutral protease expressed，and the enzyme activity of recombinant strain WB800N/pHT43-npr-PrsA increased 23%，and the enzyme activity of recombinant strain WB800N/pHT43-npr-DnaK increased 33%，compared with control strain WB800N/pHT43-npr.

Isolation，Identification and Characterization of a PCBs-degrading Bacterium

SUN Gui-ting ,CHEN Hong-yun ,ZHAO Ling-chao ,HU Xiao-ke ,CHEN Ying

2018, 34(6): 141-148. doi:10.13560/j.cnki.biotech.bull.1985.2017-1038

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This study is aimed to investigate the degradation characteristics of PCBs-degrading strain screened and isolated from estuary utilizing biphenyl as sole carbon and energy source. Pseudomonas sp. P-6-5 was investigated in more details of growth and substrate utilization pattern towards BPH and PCB3. The optimal growth conditions for strain P-6-5 were pH 7.0 and salinity 35 g/L. The use of BPH and PCB3 as an inducing agent promoted the growth of degrading bacteria. Strain P-6-5 had the transformation ability to PCB3 with substrate concentration of 10-100 mg/L. The degradation rate of PCB3 by strain P-6-5 was 95.3% in cultures containing 10 mg/L PCB3 and the maximum degradation speed was 1.9 mg/L·h. The PCBs substrate spectrum was assessed by mix13（a mixture of 13 PCB congeners）and the data showed that P-6-5 had the ability to degrade up to four chlorinated polychlorinated biphenyls. Combined with product analysis，we speculated that strain P-6-5 may have the ability to mineralize PCB3. In conclusion，Pseudomonas sp. P-6-5 has the characteristics of seawater bacteria and shows a broad range of substrate spectrum. These results postulate that the P-6-5 is an efficient PCBs-degrading strain，and has a great potential to be utilized in the bioremediation of PCBs pollution.

Screening Cellulase-producing Strains and Ion Beam Mutagenesis on Them

LIU Wan, ZHAO Shan-shan ,JIAO Zhen ,YANG Feng-yuan ,WANG Yan-ping

2018, 34(6): 149-154. doi:10.13560/j.cnki.biotech.bull.1985.2017-0810

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Seven cellulase-producing strains having large Congo red hydrolysis halo（D）and colony D/H（H is colony diameter of a strain）were isolated from soil samples containing rotten straws. Strain S-1 presented the highest CMCase activity of decomposing carboxymethylcellulose sodium，and was identified as Bacillus amyloliquefaciens. Ion beam mutagenesis was performed on S-1，and mutant strains with increased CMCase activity were screened out by high-throughput enzyme activity assay. Total 15 mutant strains with high CMCase activities after culturing for 5 generations were obtained. Among these strains，mutant strain 308，which showed totally different colony morphology，performed the highest CMCase activity. Its CMCase activity increased 84.4% after culturing for 24 h，compared to the CMCase activity of S-1；moreover，the increased CMCase activity was genetically stabile，indicating that ion beam mutagenesis has potential application value for improving the cellulase production capability of a strain.

Isolation and Identification of Bacteria Efficiently Involved in Engine Oil Degradation and Their Biodegradable Property

HUANG Man-man ,DENG Bai-wan ,WANG Meng-jiao ,CHEN Wen-qiang ,LIU Kai-hui YIN Lu

2018, 34(6): 155-163. doi:10.13560/j.cnki.biotech.bull.1985.2017-1028

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We aimed to isolate and identify highly efficient and highly-adapted engine oil-degrading bacteria for enriching the engine oil-degrading microbial strain library. Efficiently oil-degrading bacteria were obtained using the media individually supplemented with No.20 oil and vacuum pump oil as the sole carbon source，and were identified by morphology，physiological and biochemical properties，and 16S rRNA gene phylogenetics. Degradation characteristics of the strains were analyzed using UV spectrophotometry and gas chromatography mass spectrometry（GC-MS）. The most efficient oil-degrading strains，designed as JZ6，JZ18，JZ41，and JZ50，from 22 oil-degrading strains were classified as Massilia sp，Pseudomona sp，Sphingobacterium sp，and Shinella zoogloeoides sp，respectively. The degradation rate of oil reached 42.62%，33.67%，33.36% and 40.52%，when these strains were cultured in oil-containing media at 30℃ for 7 d. The strains were capable of degrading oil under the condition of pH5-9 and 20-40℃. The four strains grew on the media respectively added by dodecane，hexadecane，octadecane，benzene and naphthalene as the sole carbon source，and the strain JZ6 and JZ50 also grew well in the media containing pyrene and phenanthrene. GC-MS analysis showed that the degradation rate of strain JZ6，JZ18，JZ41 and JZ50 to total alkane was 68.66%，52.69%，49.37%，and 61.4%，and that to straight chain was 86.89%，55.98%，58.42%，and 89.13%，respectively. The strain JZ6 and JZ50 also degraded other aromatic hydrocarbon ta varied degree. The four strains of efficient oil-degrading bacteria were obtained，and had strong adaptability，thus these isolates can be used in the biological remediation of oil-polluted environment.

Effects of Manganese and Arsenic on Uranium Enrichment of Bacillus licheniformis

WANG Zi-long ,LUO Xue-gang ,SI Hui ,WANG Zhuo

2018, 34(6): 164-171. doi:10.13560/j.cnki.biotech.bull.1985.2017-0992

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This work is to understand the adsorption characteristics of uranium（U），manganese（Mn），and arsenic（As）to Bacillus licheniformis and the effects of Mn and As on the adsorption of uranium to B. licheniformis，for providing theoretical supports while applying this bacterium in the control of uranium pollution. Enrichment laws of B. licheniformis to uranium，manganese and arsenic，as well as the influences of manganese and arsenic on uranium enrichment were studied by single-factor and multiple-factor experiments. Also，the Fourier Transform Infrared Spectra（FTIR）was used to analyze the influencing mechanism. The research showed that all of them obviously inhibited the growth of B. licheniformis when the concentration of uranium，manganese，and arsenic were over 75 mg/L，25 mg/L，and 25 mg/L，respectively. Meanwhile，the concentration of uranium and manganese was negatively correlated to the adsorption rate respectively when the concentration was in 0-100 mg/L. However，the arsenic adsorption decreased at first and then rose when arsenic concentration was in 0-100 mg/L. The maximum adsorption rate of arsenic reached the highest of 79.73% while its concentration was 100 mg/L. Multiple-factor experiments suggested that arsenic promoted the uranium enrichment of B. licheniformis when its concentration was < 60 mg/L，while inhibition occurred when its concentration was > 60 mg/L. Moreover，manganese at the concentration of < 40 mg/L promoted the enrichment uranium of B. licheniformis，but inhibited when the concentration was > 60 mg/L. Manganese and arsenic affect bacteria on uranium adsorption in form of restraining bacteria growth as well as competing bacteria surface negative groups and converting properties of bacteria.

Isolation and Identification of Endophytic Microbacilli in the Gut of Periplaneta americana and Their Antibacterial Activity

LIU Ling-yan ,CHEN Zhi-yu ,ZENG Huan-xiong, LIN Pei-bin, JIN Xiao-bao

2018, 34(6): 172-177. doi:10.13560/j.cnki.biotech.bull.1985.2017-1103

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This work is aimed to investigate the morphology and molecular biology of the endophytic microbacilli in the intestine of Periplaneta americana，to study its antibacterial activity，and to analyze its potential of producing halogenated secondary metabolites at the gene level. The endophytic microbacillus in the intestine of P. americana was isolated and purified by plate scribing. After Gram staining，the cells were observed by microscopy. The 16S rDNA PCR，BLAST homology alignment and phylogenetic tree were used to identify the isolated strains in molecular biology and to screen key enzyme genes for antibiotics synthesis. Eight strains of common pathogenic bacteria were used as indicator bacteria，and the antibacterial activities were primarily tested by the method of Oxford cup. The colony colors of 8 isolated endophytic microbacilli were light yellow or white. The results of 16S rDNA PCR and BLAST homology alignment showed that the eight microbacilli had high homology with known microbacilli at 98%；while the results of phylogenetic tree demonstrated that they had high homology with the microbacilli from soil and marine. Screening of key enzyme genes in antibiotic synthesis revealed that 6 strains of above 8 presented expressions to 2 of FADH2-dependent halogenase，NRPS，and PKSI. Bacteriostatic experiments showed that 6 strains of 8 presented selective inhibitions to the growth of tested fungi and bacteria at varied level，indicating that there exist endophytic microbacilli in the intestine of P. americana.

Effects of 2，4-Epibrassinosteroid and Gibberellin A3 on the Lipid Productivity and Fatty Acid Composition of Nannochloropsis oceanica

LING Ting ,YANG Shu-ping, WEI Yu-fan, ZHANG Lin

2018, 34(6): 178-182. doi:10.13560/j.cnki.biotech.bull.1985.2017-0828

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The effects of 2，4-epibrassinosteroid（EBR）and gibberellin A3（GA3）on the growth，lipid productivity and fatty acid composition of Nannochloropsis oceanica were studied respectively. Results showed that its growth and lipid productivity were greatly enhanced with EBR of 5 and 15 mg/L，the lipid productivities increased by 7.85% and 13.97%，respectively，and the effect on total lipid content in single cell was little yet. EBR of 50 mg/L remarkably inhibited its growth. The biomass accumulation of N. oceanica was significantly improved when GA3 was each added by 5，15 and 50 mg/L in the medium. Group with 50 mg/L GA3 had the highest cell density and biomass（0.337 g/L）while its total lipid accumulation was inhibited. Overall，the lipid productivity was raised remarkably and reached 8.184 mg/（L·d）when GA3 was 15 mg/L. The proportion of saturated and monounsaturated fatty acids（16∶0，16∶1，and 18∶1）in N. oceanica decreased when adding EBR（5 and 15 mg/L）and GA3（5，15，and 50 mg/L），while the content of polyunsaturated fatty acids（18∶2，20∶4 and 20∶5）was raised significantly.

Isolation，Identification of Phosphate-solubilizing Bacteria Derived from Phosphate Tailing Soil and Their Capacities

CHEN Jia-xing ,QIN Qin ,QIU Shu-yi ,WANG Xue-li

2018, 34(6): 183-189. doi:10.13560/j.cnki.biotech.bull.1985.2017-1122

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High phosphate-solubilizing bacteria（PSBs）were isolated from phosphate tailings soil in Wengfu Group，Guizhou Province. PSBs were screened by dissolving phosphate ring test. And the genera of the phosphate-solubilizing bacteria were primarily determined by a combination of morphological characteristics identification，physiological and biochemical test，16S rDNA gene sequences analysis，and phylogenic trees. The maximum phosphate-solubilizing activity of these strains were determined by using Mo-Sb colorimetry with tricalcium phosphate as the only phosphate source，and the relationships between the abilities of different PSBs and the pH value of the culture medium were explored. The PSB1，PSB3 and PSB4 of 4 PSBs isolated from phosphate tailings soil presented obvious phosphate-solubilizing rings. The PSB1，PSB3，and PSB4 were identified to be Serratia plymuthica，Stenotrophomonas maltophilia，and Brevundimonas vesicularis，respectively. The maximum phosphate-solubilizing activities of these strains were 148.87 μg/mL，153.84 μg/mL，and 146.76 μg/mL，respectively. Compared with information in the published articles in China，the three strains all had high phosphate-solubilizing ability. For the PSB1 and PSB4，there were a significantly negative correlation between the phosphate-solubilizing ability and the pH of the culture medium，but not so for the PSB3，and the phosphate-solubilizing mechanism of the PSB3 should be further investigated.

Identification and Biocontrol Characteristics of Streptomyces sp. 30702

CONG Zi-wen ,JIAO Jing-hua ,ZHOU Shuang-qing, HUANG Dong-yi ,WU Wen-qiang ,XU Yun ,XIA We,i ZHANG Rong-ping ,HUANG Xiao-long

2018, 34(6): 190-198. doi:10.13560/j.cnki.biotech.bull.1985.2017-1083

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This work is to identify a strain of Streptomyces sp. 30702 isolated from rhizosphere of Cephalotaxus hainanensis and to estimate its biocontrol traits. According to analysis of the 16S rRNA gene sequences and multilocus sequence analysis（MLSA）as well as comparison of physiological and morphological characteristics，the Streptomyces sp. 30702 was identified as Streptomyces solisilvae，a member of the Streptomyces violaceusniger clade. The strain was able to produce protease，cellulase，chitinase，β-glucanase，1-aminocyclopropane-1-carboxylic acid（ACC）deaminase，and siderophore，and also had traits of plant growth-promoting and phosphate solubilization. Additionally，the fermented extract of strain 30702 inhibited the spore germination and mycelial growth of Colletotrichum gloeosporioides（anthracnose of yam）. The results indicate that the Streptomyces strain 30702 possesses promising biocontrol characteristics, thus it has potential development and application values in the biocontrol of plant diseases.

Effects of Total Flavonoids from Forsythia suspense on the Proliferation of Gastric Cancer Cell MGC80-3

LI Ping ,ZHANG Gui-ping, HU Jian-ran

2018, 34(6): 199-203. doi:10.13560/j.cnki.biotech.bull.1985.2017-1054

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Effects of Changzhi Forsythia suspense flavonoids（FF）on the proliferation of gastric cancer cell MGC80-3 were investigated. Total flavonoids were extracted from Changzhi F. suspense using the ethanol reflux method. MTT method，colony formation assay，RT-PCR，and Western blot were used to access the inhibitory effects of FF on MGC80-3 cell proliferation and the potential molecular mechanism. The results showed that FF significantly inhibited the proliferation and colony formation of MGC80-3 cells，and its molecular mechanism was that FF down-regulated the protein level of mTOR and up-regulated the expressions of Bax as well as cellular autophagy factor Beclin 1 and LC3 II，which promoted the autophagic cell death，then inhibiting the survival of MGC80-3 cells. Therefore, FF possesses the potential value for developing anti-tumor drug.

Screening of a Gelonin Fusion Protein with High Cell-penetrating Efficiency and Its Anti-tumor Activity and Apoptosis Pathway

ZHAI Yi-zhou ,LU Mei-ya ,ZHAO Jian ,WANG Fu-jun

2018, 34(6): 204-212. doi:10.13560/j.cnki.biotech.bull.1985.2017-1119

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By fusing gelonin with a variety of cell penetrating peptides（CPP），a recombinant protein gelonin-HBP with the highest anti-tumor activity was obtained and its molecular mechanism of inhibiting tumor cell growth was also studied. The recombinant protein of gelonin fused with four different CPPs（R9，TAT，HBD，and HBP）was expressed in Escherichia coli BL21（DE3）and then purified by NI-NTA affinity chromatography. Superhelical cleavage experiment and in vitro translation inhibition assay were used to detect the bioactivity of gelonin fusion protein. The translocation efficiency of gelonin recombinant protein was observed by laser scanning confocal microscopy. The cell growth inhibition effect of gelonin fusion protein on tumor cells was detected by MTT assay and the molecular mechanism of cell apoptosis was observed by flow cytometry and Western blot. The results showed that four gelonin recombinant proteins that retained the original biological activity of gelonin were obtained，and the fusion with a CPP promoted the cell penetration of gelonin，improved the inhibition effect of gelonin on tumor cells，and accelerated the apoptosis of these tumor cells. Compared to gelonin alone，the gelonin-HBP fusion protein increased its potency in 4 tumor cells of HeLa，B16，MCF-7 and HepG2 by 16.4 times，9.6 times，14.0 times and 24.6 times，respectively. Western blotting analysis demonstrated that gelonin-HBP induced an enhancement in the activity of Caspas 3，Caspas 8 and Caspas 9 in tumor cells，thus the expression of Bax significantly increased，while the expression of Bcl-2 significantly decreased. Conclusively，CPP may enhance the anti-tumor effect and the apoptosis of gelonin on tumor cells；among them，HBP，a CPP derived from human heparin-binding epidermal growth factor-like growth factor，makes the gelonin-HBP have the strongest anti-tumor effect. The mechanism of inducing tumor cell apoptosis by this recombinant protein is related to mitochondrial and death receptor-mediated apoptosis signaling pathway.

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2018, 34(6): 300.

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