Table of Content

01 August 2017, Volume 33 Issue 8

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Review

Study Advances on the Algal Aquaporins

LI Lu-lu, QU Chang-feng, ZHENG Zhou, WANG Yi-bin, MIAO Jin-lai, ZHANG Li

2017, 33(8):  1-6.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0152

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At present，a lot of aquaporins（AQPs）have been found in archaea，bacteria，fungi，animals and plants，but relatively few studies on aquaporins of lower plants have been carried out，especially on the algae. It is well known that there is a great difference in the growth environment between algae and terrestrial higher plants，naturally the functional mechanisms of aquaporins between them differ. The entire growth and development process of algae are closely related to water conduction，and the aquaporins in algae are not only the water transport channel but also a type of multifunctional proteins，for instance physiological and biochemical functions. Compared with the plant aquoporins，the studies on algal aquaporins were initiated later. Since the identification of the first algal aquaporin from Chlamydomonas reinhardtii in 2004，increasingly researchers began to focus on the aquaporins in algae. In recent years，on the basis of the complete genome sequences of some algae，researchers have made some new progresses in the exploration of algal aquaporins. Moreover，eight different subfamilies of algal aquaporins have been identified；and in the last two years，researchers have also found some new algal aquaporins from the Chlamydomonas sp. ICE-L，Sargassum fusiforme and Pyropia yezoensis. This paper summarizes the research advances on the classification and structure of algal aquaporins，and expounds the specific expression and physiological functions of aquaporins when the algae are in a stress environment while combined with several newly discovered algal aquaporins. This study will provide a theoretical basis for further studies on algal aquaporins

Research Progress on Male Sterility of Pepper

SHAO Gui-fang, ZHANG Fan, WANG Jiao, ZHAO Kai, MO Yun-rong, DENG Ming-hua

2017, 33(8):  7-13.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0207

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As a common and special physiological phenomenon in the plant kingdom，male sterility has high research values in basic application and practical production. In this paper，we review the pepper genic male sterility，cytoplasmic male sterility and the different abortion ways of pollen，as well as different periods of microspore abortion，and then compare some of the pollen development physiological and biochemical phenomena，mainly related to material metabolism and energy metabolism. Further，we introduce the relationship between male sterility and mitochondria from the aspects of mitochondrial metabolism，the structure and number of mitochondria，and the molecular basis of mitochondria，as well as the relevant studies on the markers and proteomics of male sterile genes using molecular technologies，so as to provide references for pepper breeding researches.

Research Progress on Breeding for Resistance to Fusarium Wilt and Powdery Mildew in Muskmelon

QIU Guo, LIU Liu, LI Xiao-mei, WANG Xue-zheng

2017, 33(8):  14-19.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0470

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Muskmelon is affected by many diseases and insect pests in the course of cultivation，among which the fusarium wilt and powdery mildew are major diseases for muskmelon. Fusarium wilt and powdery mildew are worldwide diseases，which pose a serious threat to the production of muskmelon and cause severe losses in China. Traditional agricultural control，chemical control and biological control have the problems of wasting time and effort，polluting the environment，destroying the ecological balance and endangering human health. It is not conducive to the sustainable development of agriculture. Planting disease-resistant varieties may fundamentally solve environmental and ecological problems，thus breeding disease-resistant varieties is the best solution. In this paper，we review the research progress in three aspects that include pathology，disease resistance identification and biotechnology of fusarium wilt and powdery mildew in muskmelon，and discuss the significance，problems and the development direction of disease-resistant breeding. The purpose is to provide reference for the breeding of disease-resistant cultivars of muskmelon.

Functions of Detoxification and Application Prospect of Carboxypeptidase A

XIONG Lu, WANG Xiao-yun, LIANG Zhi-hong

2017, 33(8):  20-25.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0163

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Ochratoxin A（OTA）is one of mycotoxins produced by Aspergillus and Penicillium，by which many products，such as cereals，beans，wines and coffees，are contaminated，and these contaminated foods possesses serious threats to food safety. In recent years，with the change of global climate，the food contaminated by OTA has become increasingly serious. In order to guarantee safety of humans and animals，many researchers are committing to detoxifying and degrading OTA in food and food raw materials. Currently，it is reported that OTA can be degraded by many fungi and bacteria，but few enzymes produced by them are characterized and purified，and degraded substances of OTA are unknown，thus they can’t be used in industry. Carboxypeptidase A（CPA）is an ideal product to degrade OTA because the detoxification ability of CPA to OTA is widely proved，its feature，structure and mechanism are very clear，and degraded product has no toxicity to human body；therefore，it is a novel approach to degrade OTA through genetic engineering and protein engineering to obtain a lot of stably and efficiently expressed CPA. This paper summarizes the contamination status of OTA in food，and mechanisms of bio-detoxification，the detoxification ability of CPA，and the application prospect of CPA as detoxifying agent，aiming at providing theoretical base for the development and application of mycotoxin agents degrading enzymes.

Technique and method

Research Advances on Plant Virus Detection and Virus-elimination Methods

CHI Hui-rong, MAO Bi-zeng

2017, 33(8):  26-33.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0253

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Virus is known as one of the smallest creatures in nature，its morphology and size can only be observed and identified by electron microscopy enlarging it by tens of thousands or even hundreds of thousands of times. The virus is strictly parasitic so that its growth and reproduction depend on the host cell. Plant viruses are an important part of the virus family. The plant virus disease，which affects the growth and development of plants and decreases the yield and quality，is increasingly serious year by year. Due to the numerous varieties of viruses and complex damage mechanisms，it is difficult to prevent and control the viral diseases. This article reviews the major methods of detecting plant virus as well as the technologies of virus-elimination and its application in recent years. Further，the article discusses the issues which need to be paid attention to in the virus-elimination. Finally，the article forecasts the future research direction for providing references and theoretical basis for further study.

Full-length Sequencing of 16S rRNA Gene and Its Analysis Based on the SMRT Sequencing Technology

TANG Yong , LIU Xu

2017, 33(8):  34-39.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0036

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Single-molecule sequencing，called as third-generation sequencing technology，is a high-throughput technique developed in last few years. Of them，single-molecule real-time（SMRT）sequencing technology，developed by Pacific BioSciences（PacBio），is the first commercial technology. SMRT sequencing technology could successfully overcome the disadvantage of low accuracy in the third-generation sequencing technology，by generating circular consensus sequence（CCS）through cycle sequencing the template sequence. Therefore，SMRT sequencing technology will allow scientists to profoundly and accurately study the structures and functions of microbial communities in complex environment. Here，we introduced the advantages and disadvantages of SMRT sequencing technology in 16S rRNA gene sequence of microorganism，and summarized the important steps，such as quality control，filtering of error tags，clustering analysis，annotation analysis，etc. of full-length 16S rRNA gene sequence acquired by SMRT sequencing technology. In addition，we pointed out the problems and the feasible solutions while applying SMRT sequencing technology in the study of microbial in complex environment，aiming at providing references for researchers in this field.

A Novel Approach to Establish Bio-barcode in Cells by CRISPR/Cas9

WEI Yu, ZHOU Chang-yang, LI Yan-li

2017, 33(8):  40-45.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0087

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The aim of this study is to add bio-barcode in cells by CRISPR/Cas9 for differentially marking the cells. The bio-barcode sequence，conditional induced Cas9 sequence and the corresponding sgRNA sequence were integrated into the cell through the PB enzyme，and then the cell markers were analyzed by sequencing the bio-barcode after inducing the expression of Cas9. The designed bio-barcode sequence contained 6 sgRNA target sites overlapping each other，which allowed the bio-barcode sequence to be spliced only once by Cas9. As results，the designed bio-barcode sequence in N2a cells after Dox induction was efficiently spliced by Cas9，and there were 9 different genotypes among 9 clones. After Cre inducing the E14 embryonic stem cells containing Cas9 sequence regulated by loxp-stop-loxp and bio-barcode sequence，monoclonal sequencing showed that only 2 monoclonal cells of 21 cell lines remained the original genotype，and the rest 19 strains presented 18 different genotypes respectively. In summary，this study established a novel approach to spatio-temporally add bio-barcode markers with high specificity and relative stability into cells.

Research Report

Mapping-based Cloning of a Wax Crystal-Sparse Leaf Mutant wcl1 in Rice

WANG Jia-mei, AN Xue-jiao, ZHANG Zhi-guo

2017, 33(8):  46-50.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0132

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Here a wax crystal-sparse leaf mutant（wcl1）was screened from rice T-DNA Insertion Mutant Library. The mutant wcl1has two main features as following：1）the wcl1 leaf cuticular wax reduced；2）the drought sensitivity in the wcl1 plant was higher than wild type. Map-based cloning of wcl1 revealed that a point mutation of G to T in the 10th exonof gene LOC_OS09g25850encodingketoacyl coenzymeA resulted in the termination of transcription. The analysisshowed that the wcl1was allelic to wsl2.

Cloning and Functional Analysis of Green Tissue-specific Expression Promoter of Common Wild Rice(Oryza rufipogon Griff.)

ZHAO Zhi-qiang, XUE Man-de, HUANG Ke, ZHANG Jing-wen, LONG Yan, PEI Xin-wu, YUAN Qian-hua

2017, 33(8):  51-57.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0443

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Green tissue-specific expression promoter，which makes the exogenous gene express efficiently in green tissues of receptor crop. The green tissue-specific promoter OrGSP was cloned from Oryza rufipogon，and the fusion vector of OrGSP and GUS gene was constructed and transferred into Arabidopsis thaliana in order to identify the function of the promoter. Bioinformatics analysis shows that the length of 825 bp of OrGSP，contains the basic transcription initiation elements TATA-box and CAAT-box，and light responsive elements TCCC-motif，Sp1，G-box，I-box，GA-motif and as-2-box，etc. The GUS histochemical staining of transgenic Arabidopsis thaliana shows that OrGSP regulates the expression of GUS gene only in green tissue，and the GUS activity in leaf and stem is significantly higher than in roots. The promoter of Oryza rufipogon，OrGSP，is a green tissue-specific promoter and the results can provide new regulatory elements for crop molecular breeding.

Research Report

Effects of Flor-essence on Seed Germination and Seedling Growth in Wheat

DI Hui, ZHANG Ji-quan, Lü Jian-zhou, MA Qi-yun

2017, 33(8):  58-62.  doi:10.13560/J.cnki.biotech.bull.1985.2017-0203

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The present experiment is to study the influences of different concentrations of Flor-essence on seed germination and seedling growth of wheat，in order to explore its new application in the field of plant growth regulators. By using indoor hydroponic culture，five different concentrations of Flor-essence（50，100，150，200 and 250 mg/L）were designed to determine relevant indicators on seed germination and seedling growth of wheat，and clear water（0 mg/L Flor-essence）and 0.015 mg/L brassinolide（BR）as control. As results，soaking in all different Flor-essence concentrations promoted the seed germination and seedling growth of wheat seeds while compared with the control，，and improved the seed germination potential，germination rate，germination index，root length，plant height，fresh weight，dry weight，chlorophyll content and root shoot ratio in varied degrees. Among all Flor-essence treatments，the germination conditions of seedlings were the best with the treatment of 200 and 250 mg/L；the plant height was the highest with the treatment of 150 mg/L；the root length and the root shoot ratio were the highest with the treatment of 200 mg/L；the fresh weight，dry weight and chlorophyll content were the highest with the treatment of 250 mg/L. In conclusion，the use of Flor-essence soaking promotes the seed germination and seedling growth of wheat，and the Flor-essence concentration of 150-250 mg/L may promote the seedling growth in different degree.

Cloning and Bioinformatics Analysis of Promoters and Genomic Genes of ^GmCOL1 and ^GmCOL13 in Soybean

HAN Ying-ying, CHEN Fu-lu, FU Yong-Fu, ZHANG Xiao-mei

2017, 33(8):  63-72.  doi:10.13560/J.cnki.biotech.bull.1985.2017-0201

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In the present study，the promoters and genomic genes of GmCOL1 and GmCOL13 were cloned from soybean cultivar Tianlong 1. The lengths of GmCOL1 and GmCOL13 promoters were 2 631 bp and 2 809 bp，while their genomic genes were 3 488 bp and 2 798 bp encoding 348 amino acids and 366 amino acids，respectively. Analysis by PlantCARE demonstrated that some important elements in the promoters of both GmCOL1 and GmCOL13：circadian，cis-acting regulatory element involved in light response，cis-acting element involved in ABA response，MYB binding site involved in drought-induction，and cis-acting element involved in low-temperature response. ProtParam analysis showed that GmCOL1 and GmCOL13 proteins were 38.47 kD and 40.91 kD，respectively，and both were hydrophilic. GOR analysis revealed that their secondary structure included mainly alpha helix，extended strand and random coil. Online PROSITE investigation displayed that GmCOL1 and GmCOL13 both had two zinc finger B-boxes and one CCT domain.

Type Analysis of Saponin and Gene Expression of Key Enzyme in Shanxi Soybeans

ZHAO Qiao-ling, DIAO Xiu-nan, GUO Chun-rong, ZHAO Jin-zhong, DU Wei-jun, YUE Ai-qin

2017, 33(8):  73-80.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0395

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The contents of soysaponin Aa and Ab determine the taste of soybean products，this study aims to understand the composition and content of Aa and Ab soysaponin in soybean seeds，and then to study the mechanism of anabolism and synthesis. HPLC-ESI-MS/MS was employed to measure the contents of Aa and Ab saponins in soybean，and qRT-PCR technique to study the relative expression of GmSg-1 gene in grain accumulation. Results showed among 68 Shanxi soybean seeds，the range of Aa saponin variation were 0.41-33.79 mg/g and 0.21-8.39 mg/g in hypocotyls and cotyledons respectively for Aa type soybean materials；and the range of Ab saponin variation were 0.85-44.96 mg/g and 0.26-11.09 mg/g for Ab type soybean materials. There were 19 SNP variants in GmSg-1 gene，and 10 of them resulting in amino acid substitutions were detected. The contents of Aa and Ab soysaponin increased at first and then decreased in the process of accumulation，and the contents of soysaponin were the highest after blossoming 40-50 d，while the relative expression of GmSg-1 gene was basically in the same trend as the content of soysaponin.

Isolation and Identification of Pathogenic Fungus of ^{Botrytis cinerea} and Screening of Antagonistic Bacteria Against Tomato Gray Mold

CHEN Zhe, HUANG Jing, ZHAO Jia, LIANG Hong

2017, 33(8):  81-87.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0217

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It is well known that Botrytis cinerea is one of the most geographically widespread plant pathogen on tomato. A fungus BC2016-2 was successfully isolated from the tomato surface and identified as B. cinerea，a pathogen of tomato gray mold. Then，three strains with obvious inhibitory effects were screened using B. cinerea BC2016-2 as indicator fungus，and they were Bacillus amyloliquefacien CM3，Bacillus megaterium Y-30 and Bacillus amyloliquefaciens Y-48. The greenhouse pot experiment showed that the control effects of CM3，Y-30 and Y-48 on the tomato gray mold caused by B. cinerea BC2016-2 were 65.58%，54.1% and 72.13%，respectively.

Research Report

Genome Sequencing of CMV Isolated from Pepper in Xinjiang and Polyclonal Antibody Preparation of ^CP Gene

ZHOU Dong, LIU Zhen, LIU Li, XU Peng-cheng, ZHENG Yin-ying

2017, 33(8):  88-94.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0206

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This work aims to clone gene fragment of Cucumber mosaic virus（CMV）from pepper LJ-10 in Xinjiang，to analyze the sequence，construct the prokaryotic expression vector of CMV CP gene，and to prepare CP’s polyclonal antibodies. The complete genome fragments of RNA1（3 357 nt），RNA2（3 042 nt），and RNA3（2 212 nt）were cloned by RT-PCR，and compared with different CMV isolates from CMV subgroup IA，subgroup IB and subgroup II in the GenBank by sequence analysis and phylogenetic tree analysis. The CP gene of CMV in LJ-10 was cloned into prokaryotic expression vector pET-22b and expressed in Escherichia coli BL21（DE3），and the expressed proteins were purified. Rabbit was immunized with the purified protein to prepare the antiserum with CMV-specificity，and detected by indirect ELISA test and Western blot. As results，the genomes of RNA1，RNA2，and RNA3 were successfully cloned，the sequence analysis and phylogenetic tree analysis showed that the isolates belonged to CMV subgroup IB. The prokaryotic expression vector pET-CMV-CP was successfully constructed and expressed as a 27 kD recombinant protein in E. coli BL21（DE3）by IPTG induction，and whose molecular weight was identical to the expected one. The indirect ELISA test and Western blotting showed that the antiserum’s titer was 1：500-1 000. In conclusion，the CMV isolate from LJ-10 belongs to CMV subgroup IB. The prokaryotic expression vector of CMV CP gene from LJ-10 is successfully constructed，and the corresponding polyclonal antibody is prepared.

Effects of Spermine and Spermidine on Gaseous Formaldehyde Absorption and Physiological Property in Tobacco Leaves

SI Zhi-hao, WANG Ru, FENG Yong, GUO Hong-xia, CHEN Yue, CHEN Li-mei

2017, 33(8):  95-102.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0262

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This work is to investigate the role and the mechanism of polyamine during the plants response to gaseous formaldehyde stress. With pretreatment of 100 μmol/L spermine and 200 μmol/L spermidine to model plant tobacco，the formaldehyde absorption of tobacco leaves，chlorophyll content，physiological indexes of peroxidation，formaldehyde metabolism，and the activities of the four main antioxidant enzymes（POD，CAT，APX and SOD）under gaseous formaldehyde stress were determined. The results showed that：1）The absorption efficiency of tobacco under 1，3 and 5 mg/m³ gaseous formaldehyde significantly increased by the pretreatment with spermine and spermidine，and the effect by spermine was better than that by spermidine. 2）Under gaseous formaldehyde stress，the pretreatment of spermine and spermidine delayed the degradation of chlorophyll b，and the accumulations of H₂O₂ and MDA in tobacco leaves were also inhibited. Besides，the activities of POD，CAT，APX and SOD were enhanced at varied degree with the pretreatment of spermine and spermidin under gaseous formaldehyde stress. 3）The pretreatment of spermine and spermidine presented no promotion on the metabolism of gaseous formaldehyde in tobacco.

Optimization of ISSR-PCR Reaction System on Dendrocalamus oldhami by Orthogonal Design，and Selection of Primer

XU Wen, QU Yin-quan, HAN Xiao, HE Tian-you, RONG Jun-dong, ZHENG Yu-shan

2017, 33(8):  103-110.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0004

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This work is to establish the optimal ISSR-PCR reaction system and amplifying procedure，and to screen high polymorphism primers for ISSR-PCR analysis of Dendrocalamus oldhami（Munro）. First，the genomic DNA extracted from D. oldhami（Munro）was used as template for ISSR-PCR amplification，and the orthogonal design（L₁₆（4⁵））was to investigate the optimal concentrations of dNTPs，Mg²⁺，Taq DNA polymerase，primers and DNA template，and the results were analyzed by range and variance analysis method. Then，the annealing temperature and the number of cycles were screened for establishing the optimal D. oldhami（Munro）ISSR-PCR reaction system and amplifying procedure. Moreover，the optimized system was utilized to screen the 100 ISSR primers. Ultimately，the optimal reaction system was determined as follows：in 20 μL reaction system containing 0.2 mmol/L dNTPs，2.0 mmol/L Mg²⁺，1.5U Taq DNA polymerase，0.4 μmol/L primer，60 ng DNA template，2.0 μL 10×Taq Buffer，and ddH₂O completed. The order of the effect by the factors was in Mg²⁺ > dNTPs > DNA template > Taq DNA polymerase > primer. The optimal amplifying procedure was as follows：pre-denaturing for 5 min at 94℃，38 cycles were performed with denaturing of 45 s at 94℃，annealing of 30 s due to denaturing temperature of different primer，extension of 90 s at 72℃，a final extension step of 10 min at 72℃ and stored at 4℃. Using this optimized PCR system，14 of 100 primers was selected for their clarity，high polymorphism and repetition.

Telomerase Activity in Relation to Oxidative Damage Resistance in Cells of ^{Populus euphratica} and×P. simonii P. pyramibalis cv Under Salt Stress

WU Xiao-fei, WANG Jin-yu, ZHANG Xu-yu, SUN Yu-ping, CHEN Yu-zhen, LU Cun-fu

2017, 33(8):  111-119.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0136

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Populus euphratica is an arbor that grows in drought and saline area in Northwest China. This work aims to explore the relation of telomerase activity and oxidative damage resistance. Using Populus euphratica and P. simonii×P. pyramibalis cv callus cells as materials，superoxide anion free radical levels，malondialdehyde contents and telomerase activity under salt stress were investigated. Results indicated that the fresh growth were consistent with sigmoid growth curve in both P. euphratica and P. simonii × P. pyramibalis cv cells，and the growth of P. simonii × P. pyramibalis cv was higher than that of P. euphratica. Compared to the control（0 mmol/L），the cell activity of P. euphratica increased when cultured for 7 days under the treatment of 100 mmol/L NaCl，and maintained at a certain level under the treatment of 300 mmol/L NaCl for 15 days. However，the cell activity of P. simonii × P. pyramibalis cv was affected under low NaCl concentration，and approximated to zero when cultured for 5 days under 300 mmol/L NaCl. Under the treatment of 100 mmol/L NaCl，the content of superoxide anion free radical and telomerase activity in P. euphratica cells increased compared to P. simonii × P. pyramibalis cv cells，while the malondialdehyde content in P. simonii × P. pyramibalis cv cells rose significantly. The telomerase activity in P. euphratica cells was much higher than that in P. simonii×P. pyramibalis cv cells when cultured in 300 mmol/L NaCl. The telomerase activity in P. euphratica cells increased but no significant change in P. simonii×P. pyramibalis cv cells under the low concentration of H₂O₂；high concentration of NaCl or H₂O₂ caused the oxidative damages to both P. euphratica and P. simonii × P. pyramibalis cv cells，resulting in the decrease of telomerase activity. Experimental results suggest that the telomerase can play certain role in defensing oxidative damage.

Research Report

Research on the Resistances of Several Kinds of Hydrophyte to Lead in Hydroponic Condition

JI Mei-chen, ZHANG Ji-quan, PENG Yue, MA Qi-yun

2017, 33(8):  120-125.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0199

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Hydrophytes absorbs and accumulates heavy metals and purifies the environment，therefore，owns high aesthetic and economic values. This study provided an important reference value for the selection of plant species for phytoremediation of heavy metal Pb pollution. This experiment selected 3 common hydrophytes called Iris tectorum Maxim，Acorus calamus L.，Eichhornia crassipes（Mart.）Solms to study their resistance capacities to Pb²⁺ in different concentrations of Pb（NO₃）₂ waste by using hydroponics. Then the effect of Zn²⁺ on the absorption of Pb²⁺ by water hyacinth was tested under the lead and zinc multiple-contamination. The results showed that the water hyacinth to higher concentration of Pb（NO₃）₂ waste water presented a certain resistance，in the low and middle concentration of waste A. calamus showed better resistance，and followed by I. tectorum Maxim. Under the multiple-contamination condition，synergistic effect and antagonistic effect between lead and zinc in E. crassipes（Mart.）Solms were respectively observed in the terms of uptake in different range of concentration. Different plant tissues absorbed and accumulated the Pb differently. Moreover，the heavy metal content in the aboveground of 3 hydrophytes was higher than that in the underground part. The metastasis ability of lead inside plants was not obvious，thus they do not belong to hyper accumulator. Therefore，these 3 hydrophytes are not suitable for treatment of high concentration wastewater containing lead.

Effects of Bt Cry8Ea Protein on the Midgut Microbial Flora of Holotrichia oblita Larvae

WANG Wei, ZHAO Dan, GUO Wei, LI Xin-na, ZHANG Ya-kun, YAN Xiao-ping

2017, 33(8):  126-131.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0236

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Bt Cry8Ea protein presents insecticidal activity to Holotrichia oblita larvae. The interaction between Bt Cry8Ea protein and the midgut microorganism in H. oblita larvae was investigated by metagenome DNA analysis. The midguts in normal-fed or Cry8Ea protein-fed H. oblita（2nd-instar larva）were collected and then used to extract metagenome DNAs of midgut microorganism for constructing 16S rDNA gene libraries. Polymerase chain reaction restriction fragment polymorphism（PCR-RFLP）was employed to determine the polymorphisms of 16S rDNA enzyme-spliced fragments（Msp I，Rsa I，and Hae III restriction enzyme）from the midgut microorganism of two fed larvae. Different types of clones were sequenced and then aligned with NCBI gene database，and the phylogenetic tree was constructed to study the differences of microbial communities. The results showed that there were 30 bacteria in the normal group of H. oblita，and the predominant bacteria were Desulfosporosinus（29.63%），Clostridium（12.59%），and Bacillus（10.37%）respectively. However，there were only 4 bacteria in the Cry8Ea protein-fed H. oblita group，and the predominant bacteria were Enterococcus（50%）and Streptococcus（46.88%）. These data revealed that the treatment with Cry8Ea protein changed the abundance and composition of midgut microbial flora.

Cloning and Tissue Expression Analysis of KLF3 Gene in Yak（Bos grunniens）

LIN Sen, LIN Ya-qiu, ZHU Jiang-jiang, BAI Xue, JIANG Ming-feng, WANG Yong

2017, 33(8):  132-138.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0204

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The aims of this study are to clone yak KLF3 gene sequence，to analyze its biological characterization，and to clarify its expression patterns in different tissues. Six healthy，4-6 years old male Maiwa yaks were selected as experiment animals. After fasting slaughter，the tissue samples of spleen，lung，kidney，subcutaneous fat and longissimus dorsi muscle were collected for the total RNA extraction. The Reverse Transcription PCR（RT-PCR）was used to clone Maiwa yak KLF3 gene sequence，the quantitative real-time PCR（qPCR）was used to detect the expression levels of KLF3 gene in different tissues. The results showed that the sequence of yak KLF3 gene was 1 137 bp（GenBank accession number：KX964630），containing 963 bp CDS，32 bp 5' UTR and 64 bp 3' UTR，encoding 346 amino acids. The amino acid sequence of yak KLF3 shared a similarity of 100% with that of cattle（XP_010820342.1）. The mRNA expression level of KLF3 was high in lung and liver tissue，significantly higher than other tissues（P < 0.01）.

Research Report

Promoter-independent Transcription of Exogenous DNA and Subsequent RNA Splicing in Mammalian Cells

XU Wen, YANG Hua-yu, ZHENG Yong-chang

2017, 33(8):  139-145.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0183

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Introduction of exogenous double stranded DNA into mammalian cells will undergo restructuring，rearrangement and other complex molecular and biochemical reaction. Few DNA molecules will randomly integrate into the host genome，namely the occurrence of horizontal gene transfer. However，it is unclear whether these exogenous DNA molecules can be recognized by cell machine and triggers the transcription in the circumstance of containing no special promoter. During the construction of circle RNA（circRNA）expression system，we discovered that promoter-independent transcription of exogenous DNA occurred，also RNA splicing was in accord with “GU-AG” in transcripts，and subsequently abnormal spliced molecules similar to the circRNA formed. Further studies showed that the promoter-independent transcription and splicing of exogenous DNA in mammalian cells were conservative. Conclusively，this work revealed a new destiny approach of exogenous DNA in the mammalian cells，which is of significance and applicability in studying the effects of exogenous DNA on host cells.

Acid Domestication and UV Induction Increase Microalgae Tolerance to Acid Stress

SHI Fei-fei, CHEN Tong, CHENG Wei-lan, SONG Cheng-Fei, JI Chun-li, LI Run-zhi

2017, 33(8):  146-151.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0446

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Utilization of microalgae for direct fixation of carbon from coal-based flue gas may reduce CO₂ emissions，and also produce bio-based products with multiple values. However，CO_2，NOx and SO₂ at high concentration in the flue gas always cause excessive acidification of culture medium，leading to severe inhibition of microalgae growth. The present study was conducted to establish a simple and effective method of microalgae domestication for enhancing the microalgae resistance against acid stress and increasing their initial growth rate when inoculated in low-pH medium. The initial growth rate of the acid-domesticated and the UV-treated Scenedesmus reached 0.253 g/（d·L），which was significantly higher than that（0.132 g/（d·L））by only acid domestication at pH4.5 and that（0.092 g/（d·L））of 5-min induction at low-dose of UV radiation in low-pH environment. The more remarkable effects were achieved by combining such acid domestication and UV induction. The domesticated seed algae grew normally in the acidic environment of pH3，but the non-domesticated microalgae growth stopped. The initial growth rate of the domesticated seed algae was 6.6 times higher than that of the non-domesticated microalgae in low-pH environment. The above results revealed that acid domestication and UV induction significantly improved Scenedesmus tolerance against low-pH environment.

Function of Mef2 in Mitochondria of ^{Schizosaccharomyces pombe}

ZHANG Juan, CHEN Mei-xiu, SHANG Jin-jie

2017, 33(8):  152-158.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0097

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Mitochondrial translation，essential for synthesis of the electron transport chain complexes in the mitochondria，is governed by nuclear encoded gene. This paper aims to reveal the main function of Mef2 protein in Schizosaccharomyces pombe. First，we constructed the strain of mef2 gene deletion by homologous recombination，and observed its growth phenotype in non-fermentative medium with glycerol as the sole carbon source. Second，the bioinformatics analysis demonstrated that the N-terminal of Mef2 contained a mitochondrial localization sequence（MTS）consisting of 31 amino acids. To further determine the localization of Mef2 protein，a GFP fluorescent label was added to the C-terminus of Mef2 to observe GFP green fluorescence. Third，Northern blotting was used to detect the effect of mef2 deletion on the level of mitochondrial genome encoding mRNAs. Finally，Western blotting was employed to detect the influence of the deletion of mef2 on the expression of mitochondrial genome-encoded protein. The results showed that Δmef2 strain caused growth defects on non-fermentation medium，which was one with mitochondrial respiratory defect. GFP green fluorescence localization experiments confirmed that Mef2 was localized in mitochondria. Northern blotting revealed that the deletion of mef2 did not affect the transcription of mRNAs encoded by the mitochondrial genome. Western blotting showed that the deletion of mef2 resulted in decrease in the expression of Cox1，Cox3，Atp6，and Cob1 proteins. In summary，Mef2 is a protein that is closely related to mitochondrial function and is involved in the translation of mitochondrial encoded proteins Cox1，Cox3，Atp6，and Cob1.

Enhanced Production of Vitamin B₁₂ by Screening the Promoter in Pseudomonas denitrificans

WANG Ling-ling, Xia Miao-miao, DONG Hui-na, ZHU Bei-wei, ZHANG Da-wei

2017, 33(8):  159-166.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0010

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Vitamin B₁₂（VB₁₂/Cobalamin）possesses several physiological functions and is widely used in pharmaceutical and food industries. Pseudomonas denitrificans is commonly employed in the production of VB₁₂. In order to improve the VB₁₂ productivity by a strain，the high-expression promoters were screened and then used to express the gene synthetizing VB₁₂. Via online prediction software，analyzing the promoters in promoters-included non-coding sequences that precede the genes encoding heat shock protein and molecular chaperone in P. denitrification，the non-encoding sequences of P_ibpA，P_cbpA，P_dnaJ，P_htpG，P_dnak，and P_grpE were selected and ligated to GFP report gene encoding green fluorescent protein. Then the GFP fluorescence signal value was detected by enzyme standard instrument，further for obtaining the promoters with high-expression，the expression levels of promoters in the non-coding sequences preceding the genes encoding heat shock protein and molecular chaperone were evaluated，which then was applied for the overexpression of genes in the VB₁₂ synthetic pathways . Fluorescence experimental results showed that the expression of GFP fluorescence value for the non-coding sequence with the promoter P_dnak was the highest. Subsequently，the strongest P_dnak promoter was selected to construct the recombinant P. denitrificans overexpressing gene cobA in VB₁₂ synthesis pathway，and the fermentation results revealed that the VB₁₂ yield increased by 21.5mg/L compared with the control strain. Conclusively，screening high-expression promoter for overexpressing the key gene in VB₁₂ synthesis pathway is an effective approach for improving VB₁₂ production.

Research Report

Fermentation Conditions of Antagonistic Bacterium Pb-4 Against Fusarium Wilt Using Soybean Processing Wastewater

GUO Jun, WU Ai-lian, YAN Min, PANG Jin-mei, JIAO Xiao-yan

2017, 33(8):  167-173.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0159

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The wastewater from soybean processing（yellow serofluid）was used to optimize the fermentation medium condition of antagonistic bacterium Pb-4 against fusarium wilt. The single factor experiments were conducted to screen the carbon source of Pb-4 strain in yellow serofluid medium. Orthogonal experiments L₁₈（3⁵）were designed to select the optimal carbon source and inorganic components for Pb-4 strain in yellow serofluid medium，and determine the optimal fermentation medium formulation. The fermentation temperature，pH and inoculation amount of Pb-4 strain were optimized by response surface method（RSM）. As results，the composition of the optimal medium was yellow serofluid 1 000 mL，corn flour as the optimal carbon source 30 g，（NH₄）₂SO₄ 2 g，MgSO₄ 1.5 g，CaCO₃ 1.5 g and FeSO₄ 0.3 g. The fermentation conditions after optimization were as：temperature 36.3℃，inoculum size 5%，pH 7.4 and incubation time 40 h. Under the optimized conditions，the number of fermented bacteria was up to 42.8×10⁸ CFU/mL_，and the maximum biomass OD₆₆₀ was 0.393，which was in accord with the predicted value 0.390. In conclusion，the optimized medium can be as an effective substitute for traditional medium like peptone，yeast powder，glucose，etc.，moreover as it is simple，cost-efficient and environmentally friendly，thus it can be used as raw material for the scale production of microbial agents.

Effects of S-malonyltransferase Gene Inactivation on Rifamycin Production by Amycolatopsis mediterranei

HE Si-yu, CHENG Wen-yu, JIN Hong-xing

2017, 33(8):  174-179.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0200

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To increase rifamycin yield，the Amycolatopsis mediterranei mutant of the inactivated S-malonyltransferase gene（fabD）was constructed. S-malonyltransferase gene homologous recombinant vector was constructed using fusion PCR，and then transformed into A. mediterranei by electroporation，making the homologous recombination occurr. Then fabD-inactivated strain was screened by apramycin as a marker. Further，the rifamycin production between mutant strains was wmpared and the parental strain by fermentation. The results showed that the homologous recombinant vector of fabD was successfully constructed，and the A. mediterranei △fabD was obtained；the production of rifamycin SV in the mutant strain was 168.08 mg/L，with 9.94% increase in contrast to the parent strain. The inactivation of S-malonyltransferase gene weakened the fatty acid synthesis of mutant strain，subsequently strengthened the synthesis of rifamycin.

Research Report

Site-directed Mutation of α-ketoglutorate Semialdehye Dehydrogenase and Its Enzymatic Properties

QIN Hai-bin, XIONG Tao, ZHANG Bo, NIU Kun

2017, 33(8):  180-185.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0188

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3-HP is an excellent chemical intermediate，and the study on the production of 3-HP with glycerol as substrate is favored，while the main reason for limiting 3-HP production is the low activity of AldH. A gene of α-ketoglutorate semialdehye dehydrogenase（KGSADH）from Azospirillam brasilense was constructed by homology modeling and structure analysis. The KGSADH with high activity was obtained by site-directed mutation. The KGSADH was expressed and purified for the investigation of enzymatic properties. The results revealed that the activity of TU-KGSADH（E120D / P219A）reached 6.03 U/mg，which was 322% higher than that of the original KGSADH. The optimal temperature of TU-KGSADH decreased from 35℃ to 30℃，the thermostability reduced，and the optimal pH was 8.0 and enzymatic activity was improved slightly under pH 6.0-8.0. Zn²⁺ had a strong inhibitory effect on the activity of KGSADH，while Co²⁺，Fe³⁺ and Fe²⁺ had a great effect on the activity of KGSADH. In the kinetic aspect of the TU-KGSADH，the Km with acetaldehyde as a substrate reduced from 7.58 to 6.28 mmol/L，and Vmax increased from 10.6 to 12.3 U/mg. Finally，the factors affecting the changes of enzymatic properties were analyzed，The 120 site mutation was associated with the decrease of the optimal pH and pH stability to acidic migration，and the 219 site mutation may be the reason for the decrease of the optimum temperature and thermal stability，providing a fine reference for further research in directional modification of aldehye dehydrogenase.

Recombinant Expression of NADPH-Dependent Mannitol Dehydrogenase and Transformation Conditions of Mannitol

FENG Zhi-mei, ZHAO Ya-tong, LIU Ye-xue, LU Fu-ping, LI Yu

2017, 33(8):  186-191.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0168

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This work is to analyze the heterogeneous expression of NADPH-dependent mannitol dehydrogenase and the transformation of fructose for laying a foundation of constructing mannitol synthetic way. The recombinant strain BL21（DE3）/pET28a-mdh of expressing mannitol dehydrogenase was constructed and induced，and the target protein was purified with His-tag. Mannitol production from mannitol dehydrogenase transforming fructose was analyzed by HPLC. As results，the recombinant strain was constructed successfully and the mannitol dehydrogenase was purified. The activity of the purified mannitol dehydrogenase was 270 U/mL. The conditions of fructose transforming to mannitol by purified mannitol dehydrogenase were optimized，and the optimal conditions were as follows：300 g/L substrate fructose，pH5.8，9 mmol/L NADPH and incubation at 40℃. Under the optimized conditions，the transformation rate of mannitol reached 97.4%. In conclusion，the NADPH-dependent mannitol dehydrogenase was expressed successfully in Escherichia coli，providing the basis for studying the metabolism regulation of mannitol synthesis in E. coli.

Optimization of the Thermal Activity and Stability of Hyperthermophilic α-amylase ApkA

ZENG Jing, GUO Jian-jun, YUAN Lin, YANG Gang, CHEN Jun

2017, 33(8):  192-198.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0172

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This work aims to obtain α-amylase with improved thermal activity and stability. Based on the structure analysis of hyperthermophilic α-amylase ApkA and the most thermo-stable α-amylase PFA，a Zn²⁺-binding site mutant ApkAdsK152H/A166C was constructed by introducing the Zn²⁺-binding site of PFA into ApkA. The mutant ApkAdsK152H/A166C exhibited effective increase in terms of thermal activity and stability. The optimal temperature of the mutant increased from 90℃ to 100℃ and the corresponding specific activity was 5 201.08 U/mg. The half-life of ApkAdsK152H/A166C prolonged from 5 h to 10 h while incubated at 90℃，and from 7.5 min to 80 min at 100℃. The results of Zn²⁺ content measurement in recombinant α-amylases confirmed that ApkAdsK152H/A166C bound with one Zn²⁺ ion. These results suggested that the introduction of Zn²⁺-binding site improved the thermal activity and stability of ApkA.

Study on Inhibition Mechanism of Melanogenesis and Melanin Synthesis of B16F10 Cell Induced by Two Natural Products

CHENG Xing-an, ZHANG Shu-ming, ZHOU Xiao-wu, WU Bo, LIN Xian-wei, QIN Xiang-jing, HUANG Su-qing, LIU Zhan-mei, JIANG Xu-hong

2017, 33(8):  199-205.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0122

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This study aims to clarify the inhibition mechanism of proliferation and melanin synthesis of B16F10 cell induced by 10-hydroxycamptothecin（HCPT）and resveratrol（Res）. MTT cytotoxicity assay，microscope observation-assay，L-Dopa oxidation-assay and NaOH cracking-assay were employed to investigate the proliferation，cellular morphology，tyrosinase（TYR）activity and melanin synthesis of B16F10 cells treated by HCPT and Res at different concentrations，respectively；and semi-RT-PCR to analyze the relative mRNA expression of TYR and microphthalmia associated transcription factor（MITF）in B16F10 cells. It was found that HCPT（40，80，120，160，and 200 μmol/L）and Res（80，120，160，and 200 μmol/L）inhibited B16F10 cell proliferation through inducing apoptosis，and exhibited significantly dose-dependent inhibiting effects on tyrosinase activity and melanin synthesis on B16F10 cell（P < 0.05）. In addition，relative mRNA expressions of the TYR and MITF genes in B16F10 cells were depressed remarkably by HCPT at different concentrations and Res at high concentrations（120 and 160 μmol/L）. Conclusively，HCPT and Res may inhibit the B16F10 cell proliferation via inducing cell apoptosis，and depress the melanin synthesis in B16F10 cells from down-regulating the transcription of MITF gene and subsequently inhibiting the gene expression of TYR mRNA and activity of TYR.

Interaction Analysis of Thyroid Hormone Receptor with Drosha Protein

SHANG Pan, FU Yuan-shuai, SHI Zhi-yi, YU Jie, LIU Su-ping

2017, 33(8):  206-212.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0174

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This study isto demonstrate the interaction between the thyroid hormone receptor（THR）and the Drosha protein. Selecting HEK293 cells as materials，the protein immune complexes were acquired by co-immunoprecipitation method，and then detected by Western blot and analyzed by mass spectrometry. The results showed that there was THR protein detected by THR antibody Western blot，and Drosha protein was detected by Drosha antibody Western blot. Concurrently，the Drosha protein was identified by mass spectrometry after immunoprecipitation with THR antibody，and the mass spectrometry after Drosha antibody immunoprecipitation confirmed that therewas also a THR protein，suggesting that there was interaction between the THR and the Drosha protein. Moreover，the Go cluster analysis for the primarily screened 54 proteins interacting with both THR and Drosha protein revealed that the identified proteins were mainly composed of cell membrane and nucleus，and they were involved in varied biological pathways such as cell migration，cell apoptosis，protein metabolism，immune responses，signal pathways and transcription regulations，etc.，also had molecular functions of binding with nucleic acid and cytoskeleton.