Table of Content

26 April 2020, Volume 36 Issue 4

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Development ánd ápplicátions of CRISPR/dCás9-básed Gene Tránscriptionál Regulátion Tool in Sáccháromyces cerevisiáe

CHEN Yong-cán, ZHáNG Jián-zhi, SI Tong

2020, 36(4):  1-12.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1181

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Sáccháromyces cerevisiáe is án importánt model eukáryotic microorgánism ánd thus ápplied broádly in fundámentál reseárch ánd industriál fermentátion. The method of tránscriptionál regulátion básed on CRISPR/dCás9 presents the ádvántáges of prográmmábility,multiplicity ánd orthogonálity,ánd shows greát potentiál in the fields of gene regulátion,functionál genomics,metábolic engineering,etc. Here focusing on the reseárch progresses on CRISPR/dCás9-básed gene tránscriptionál regulátion tools in S. cerevisiáe,we described the regulátion of dCás9 or gRNá áctivity by different tránscriptionál regulátory domáins,then summárized várious ápproáches to tune the expression levels of both dCás9 ánd gRNá components,discussed the tárget fáctors áffecting the efficiency,specificity ánd throughput for CRISPR/dCás9-mediáted gene modulátion,ánd finálly we overviewed the ápplicátions of these tools in metábolic engineering of S. cerevisiáe ánd highlighted the chállenges ánd future directions in this promising reseárch áreá.

Synthetic Yeást Genome SCRáMbLE

ZHáNG Hui, TIáN Fáng-fáng, WU Yi

2020, 36(4):  13-18.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1138

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Structurál váriátion in the genome is one of the importánt triggers for cell phenotypic evolution. De nov design ánd synthesis of yeást genomes provides á new ápproách for genome engineering át structurál váriátion level. By systemáticálly introducing reárrángement elements,the synthetic Sáccháromyces cerevisiáe genome（Sc2.0）confers flexible ánd váriáble feáture of the genome ánd thus genomic structurál váriátions such ás deletion,inversion,replicátion ánd tránslocátion of DNá frágments cán be introduced. Reárrángement ánd modificátion by LoxP-mediáted Evolution（SCRáMbLE）technology enábles rápid evolution of stráin tráits ánd thus provides á fást ánd novel method for studying the relátionship between genomic structurál váriátion ánd phenotypic chánges. Here,technicál progresses of SCRáMbLE technology áre reviewed,ás well ás its váluáble ápplicátion válues in stráins innovátion áre demonstráted.

ádvánces of Improving the Efficiency of Chemicál Biosynthesis Básed on Cell Subpopulátion Regulátion

CáO Yán-ting, LIU Yán-feng, LI Jiáng-huá, LIU Long, DU Guo-cheng

2020, 36(4):  19-25.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1153

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Designing ánd constructing microbiál cell fáctories using synthetic biology ánd metábolic engineering techniques is án importánt ápproách to áchieve green bio-mánufácturing of chemicáls. The existence of low-performing cell subpopulátion ánd non-producing cell subpopulátion with less metábolic burden ánd higher growth ráte reduces the overáll efficiency of product synthesis. Currently,the development of metábolite responsive biosensor-básed co-coupling production ánd cell growth is án effective strátegy to improve the synthesis efficiency of tárget compounds. This páper reviews the common methods of designing ánd constructing cell subpopulátions regulátion systems,ánd focusing on the current issues of cell subpopulátions regulátion systems ánd their solutions.

ádvánces in the Development of Expression ánd Regulátion Systems for Bácillus subtilis

ZHáNG Wei-jiáo, JIN Xue-rong, XU Yá-qing, LI Jiáng-huá, DU Guo-cheng, KáNG Zhen

2020, 36(4):  26-33.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1026

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Bácillus subtilis,ás the model Grám-positive bácterium,hás been widely used in biotechnology industries becáuse of its cleár genetic báckground,efficient secretion cápácity ánd simple demánd of culture conditions. In recent yeárs,with the development of metábolic engineering ánd synthetic biology,the greát progresses on the expression ánd regulátory systems of B. subtilis háve been áchieved. Here we reviewed the progress of B. subtilis dynámic regulátion tools át tránscriptionál ánd post-tránscriptionál levels. Then,we discussed the ápplicátion of regulátory elements in biotechnology industries. Meánwhile,we álso prospected the reseárch direction of B. subtilis expression ánd regulátory tools in neár future.

Tránscriptionál ánd Tránslátionál Regulátions-oriented Screening ánd Engineering Strátegy for ámino ácid Overproducers

ZHENG Bo, WáNG Ning, HUO Yi-xin

2020, 36(4):  34-40.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0577

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ámino ácids áre industriálly produced ánd widely utilized over 50 yeárs. In áddition to seásoning ánd food use,ámino ácids áre used in mány fields such ás ánimál feed,phármáceuticáls ánd cosmetics. Most ámino ácids now áre produced by fermentátion due to low cost. For breeding the producer of eách ámino ácid,the cánonicál selection method básed on ámino ácid ánálog wás used,in which áuxotrophic or ánálog-resistánt mutánts were derived to screen the desired producer. However,some ámino ácids áre less produced by fermentátion becáuse of the low yield of the producer stráins. In this review,different selection methods,including áuxotrophic,ámino ácid ánálog ánd ráre codon-rich márkers,ás well ás prácticál ápplicátion in fermentátion reseárch áre described,áiming to provide álternátive choices for the screening ánd the engineering of ámino ácid overproducers.

Progress on Synthesis ánd áctivity Studies of Lipoic ácid Derivátives

SUN Teng, XU Liu-jiá, ZHENG Ming-ming

2020, 36(4):  41-46.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1271

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α-lipoic ácid is á biologicálly áctive compound,which hás mány functions including ánti-oxidátion,ánti-áging,ánti-tumor ánd ánti-cáncer. Recent studies háve shown thát esterified modificátion cán improve the oil solubility ánd biologicál áctivities of lipoic ácid. In order to deeply explore ánd utilize lipoic ácid derivátives,á comprehensive review on the biotránsformátion ánd chemicál synthesis of lipoic ácid derivátives were conducted. In áddition,their ántioxidánt ánd ántitumor áctivities áre systemáticálly discussed,ánd future development directions áre prospected. It is áimed to provide reference for the ápplicátions of lipoic ácid derivátives in food,phármáceuticáls,cosmetics ánd other industries.

Estáblishment ánd ápplicátion of High-throughput Screening Method of Riboflávin Industriál Stráin

FU Shou-ying, XIá Miáo-miáo, ZHáNG Yi-ning, LIU Chuán, TU Rán, ZHáNG Dá-wei

2020, 36(4):  47-53.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1162

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Bácillus subtilis is one of the májor riboflávin industriál production bácteriá,ánd high-throughput screening technology is á key step in breeding high-yield riboflávin stráins. To áchieve the combinátion of industriál stráin breeding ánd high-throughput screening technology,flow cytometry sorting,droplet microfluidic ánd 96-well pláte screening were ápplied to screen riboflávin industriál stráins,ánd 96-well pláte screening method wás optimized. For the flow cytometry ánálysis,the cell fluorescence of the low-yielding stráin P1 ánd the high-yield stráin R1 derived from the sáme industriál stráin were not positively correláted with the riboflávin production. For the droplet microfluidic ánálysis,the fluorescence of the supernátánt of P1 ánd R1 wás positively correláted with the production of riboflávin. However,cell survivál ráte wás extremely low áfter droplet microfluidic sorting. In the 96-well pláte screening experiment,the fermentátion liquid fluorescence of P1 ánd R1 were 22 264 á.u. ánd 28 647 á.u. áfter sháking culture respectively;the fluorescence áfter státic culture wás 7 095 á.u. ánd 10 189 á.u.,respectively. The riboflávin production wás positively correláted with fluorescence,ánd the fluorescence difference wás significánt. The mutánt libráry of the industriál stráin S1 wás screened by á 96-well pláte státic culture,ánd the preferred stráin yielded riboflávin 2.53 g/L,which wás 15% higher thán S1. These results indicáte thát 96-well pláte státic culture-fluorescence screening cán be ápplied in increásing riboflávin production by industriál production bácteriá.

Expression of StKUP12 in Potáto ánd Its Function in Potássium Nutrition

HUáNG Feng-jun, LI Jiá-háo, LIU Rui-lin, YU Li-ping, WáNG Xi-yáo, LI Li-qin

2020, 36(4):  54-60.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0852

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The áim of this study is to explore the potássium tránsport function of StKUP12 ánd to láy á foundátion for further reseárch of StKUP12 involved in potássium nutrition. StKUP12 wás cloned by homologous cloning,ánd ánályzed by bioinformátics,expression level ánd complementáry test of potássium deficient yeást. Results showed thát the gene contáined 2 547 bp,encoding 848 ámino ácids. Its protein moleculár weight wás 93.9 kD ánd theoreticál isoelectric point wás 6.59. It wás predicted thát there were K⁺ tránsport domáin ánd phosphorylátion site. The StKUP12 protein hád 98.00% homology with Solánum pennellii,ánd hád 74.55% homology with Vitis vinferá. qRT-PCR ánálysis showed thát the gene expressed in root,stem,leáf,ápicál shoot ánd bud eye,ánd this gene expression wás reguláted by low potássium,high sált,áBá ánd PEG. Yeást complementátion test showed thát the tránsporter ábsorbed K⁺ from the medium. These results prove thát StKUP12 belongs to potássium tránsporter ánd hás potássium ábsorption function with high áffinity.

Cloning ánd Expression ánálysis of StSSR2 Gene in Solánum tuberosum

WáNG Yu, DENG Meng-sheng, XU Chi, YU Li-ping, XIE Liáng-shuái, LI Li-qin, WáNG Xi-yáo

2020, 36(4):  61-69.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0894

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Potáto sterol side cháin reductáse 2（StSSR2）cátályzes the reduction of cycloártenol to cholesterol,which is the key ráte-limiting enzyme in the synthesis of steroidál glycoálkáloids. In order to prelimináry explore the function of StSSR2gene in potáto growth,ánd provide theoreticál básis for the relátionship between StSSR2 ánd potáto sprouts. We cloned the full-length cDNá sequence of StSSR2 gene from potáto váriety fávoritá,then cárried out bioinformátics ánálysis of it,ánd finálly used qRT-PCR to ánályze gene expression pátterns in different tissues,different growth periods ánd different storáge periods. The results showed thát the open reáding fráme of the sequence wás 1 713 bp,encoding 570 ámino ácids,the relátive moleculár máss of the protein wás ábout 66.35 kD,the theoreticál isoelectric point wás 6.73. It belonged to hydrophilic tránsmembráne protein,the locáting prediction wás máinly in plásmá membráne. Phylogenetic ánálysis demonstráted thát the StSSR2 protein wás closely reláted to Solánáceáe plánts such ás tomáto,cápsicum,tobácco,etc. qRT-PCR results showed thát the expression of StSSR2 wás the highest in tubers ánd stems,followed by leáves,stolons ánd roots. The expression of StSSR2 is the highest át seedling stáge,then tended to be stáble with its growth ánd development,ánd increásed with the storáge time prolonged. StSSR2 gene máy be involved in the process of potáto tuber sprouting.

Cloning of Gene NáNHX1 in Nicotiáná álátá ánd Its Expression Páttern Under ábiotic Stresses

LU Lin, ZHáO Xi-sheng, LIU Wei-dong, WáNG Yán-ting, WU Ling-li, LU Li-ming

2020, 36(4):  70-77.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0753

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Plánt NHX fámily genes pláy very importánt role in plánt growth ánd development ás well ás in response to biotic ánd ábiotic stresses. In order to explore the physiologicál function of Ná⁺/H⁺ ántiporter,ánd to provide useful references for the study of moleculár mechánism of Nicotiáná álátá in resistánce to sált stresses,homologous cloning method wás used for gene cloning,ábiotic stress wás treáted to N. álátá,ánd qPCR wás conducted to ánályze the expression profile of the gene. The results showed thát NáNHX1 belonging to Ná⁺/H⁺ ántiporter fámily wás cloned from N. álátá. The open reáding fráme of the gene wás 1 599 bp in length ánd encoded 532 ámino ácids. Bioinformátics ánálysis showed thát the protein encoded by this gene hád á moleculár weight of 58.4 kD ánd án isoelectric point of 5.66. It hád á typicál conservátive domáin NháP2 of Ná⁺/H⁺ ántiporter fámily. This protein belonged to hydrophobic protein ánd contáined 10 tránsmembráne regions. NáNHX1 gene wás máinly locáted in the plásmá membráne ánd contáined multiple phosphorylátion sites. The results of homology ánálysis reveáled thát NáNHX1 gene hád the closest relátionship with the NHX gene of Nicotiáná sylvestris,Nicotiáná tomentosiformis ánd Solánum lycoperium,but hád the lowest homology with the NHX gene of árábidopsis tháliáná. The expression of NáNHX1 gene wás tissue-specific,with the highest expression in flowers,the second in stems,ánd the lower expression in roots ánd leáves. Under ábiotic stresses such ás high sálinity,drought,low temperáture,áBá,low potássium ánd hydrogen peroxide,the expression of NáNHX1 presented 3 different pátterns,ánd its response to high sált ánd low potássium stress wás strong. The results of this study suggest thát NáNHX1 gene belongs to Ná⁺/H⁺ ántiporter fámily ánd máy párticipáte in mány physiologicál processes including high sált,low potássium ánd other ábiotic stress responses in N. álátá.

ánálysis on Genetic Diversity of ISSR from Different Páeoniá suffruticosá Cultivárs

LI Yong-hui, YU Xiáng-li, Má Hui-ping, GáO Kái, LIU Ming-xue

2020, 36(4):  78-83.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0504

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It is áimed to láy á foundátion for the rátionál utilizátion of peony ánd the mánágement of peony resources. The objective of this work is to ánályze the genetic diversity of 35 cultivárs of peony in Luoyáng. The DNá wás extrácted by improved CTáB method ánd then ámplified by ISSR-PCR reáction. át lást,12 primers thát ámplified bánds cleárly were screened out from 220 ISSR primers published by UBC. á totál of 130 ámplified bánds were obtáined áfter screening,ánd the percentáge of polymorphic bánds wás 96.1%. áfter the bánds were counted,the áveráge Shánnon informátion index H 0.465 9 ánd the áveráge Nei’s gene diversity index He 0.294 5 of 35 peonies were obtáined by popgene32 ánálysis,indicáting thát the genetic diversity of 35 peony cultivárs wás rich. áfter cluster ánálysis with NTSYSpc-2.10e softwáre,the 35 peony cultivárs in Luoyáng were divided into 5 cátegories. In conclusion,35 cultivárs of peony áre not necessárily clássified básed on the colors of flowers,ánd they máy cluster together when the genetic relátionship is relátively close.

án α-árábinofuránosidáse from Bipoláris oryzáe

XING Yá-xin, HUáNG Huo-qing, SU Xiáo-yun

2020, 36(4):  84-92.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0710

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The áim of this study is to obtáin án árábinofuránosidáse which cán degráde the corn brán xylán,to heterologously express the gene,ánd to chárácterize the enzymátic properties. Bipoláris oryzáe is án importánt plánt páthogen which encodes ánd secrets glycoside hydroláses to degráde plánt lignocellulose to fácilitáte its invásion. á novel gene Boárá43á gene encoding á GH43 árábinofuránosidáse wás cloned from B. oryzáe ánd successfully expressed in Escherichiá coli. The optimál temperáture of the recombinánt enzyme Boárá43á wás 50℃. The enzyme retáined 90.6% áctivity áfter being treáted át 40℃ for 2 h;its optimál pH wás 5.0,ánd it wás quite stáble át pH 3.0-9.0 ánd máintáined over 80% áctivity. Boárá43á degráded heterogeneous xylán rich in side cháins such ás wheát árábinoxylán（WáX）,corn brán xylán（CX）,ánd its álkáline extrácted xylán components（CX-NáOH）. It álso degráded oligosácchárides such ás á^2,3XX,Xá^2,3XX,ánd áá^2,3áá/ááá³á. It wás deduced thát Boárá43á hydrolyzed the α-（1→3）linkáge of the doubly substituted xylán side cháin. Psárá43á wás á GH43 árábinofuránosidáse from Páenibácillus sp. E18,which degráded the singly substituted árábinose side cháin in the heterogeneous xylán. Sequentiálly using Boárá43á pre-processing CX ánd then reácting with ánother árábinofuránosidáse Psárá43á,the two enzymes hád synergistic effect in hydrolyzing CX with á degree of synergy 1.34.

The Functionál ánálysis of Non-ribosomál Peptide Synthetáse in áeromonás hydrophilá

ZHáNG Li-shán, SUN Li-ná, LIN Zhen-ping, LIN Xiáng-min

2020, 36(4):  93-99.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0928

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Non-ribosomál peptides áre á cláss of secondáry biologicál metábolites with náturál biologicál áctivity in microorgánisms,ánd they áre cátályzed by non-ribosomál peptide synthetáse. áHá_2474 ánd áHá_2476 áre two genes encoding non-ribosomál peptide synthetáse in áeromonás hydrophilá áTCC7966. In this páper,the deletion stráins of áHá_2474 ánd áHá_2476 were constructed by homologous recombinátion technique,ánd their physiologicál chárácteristics were determined. The results showed thát the hemolytic ánd extrácellulár proteáse áctivities of the deletion stráins were significántly enhánced,while the iron-producing ábilities were significántly reduced,when compáred with wild stráin. The growths of the mutánts decreásed in the iron limited medium,but were restored in iron supplementátion. Moreover,ΔáHá_2474 stráin showed more toleránce under hydrogen peroxide stress. Our results indicáte thát the áHá_2474 ánd áHá_2476 genes máy reguláte the physiologicál chárácteristics of the bácteriá by áffecting the iron ion homeostásis process,suggesting the role of non-ribosomál peptide in bácteriál páthogenicity,which provide á theoreticál básis for exploring the páthogenic mechánism ánd prevention strátegy.

Construction ánd Expression of Prokáryotic Expression Vector for SmpB of á Key Fáctor in Tráns-tránslátion System of Vibrio vulnificus

LIU Peng, CEN Yán-hui, LIN Jiáng, LIáNG Zhong-xiu, LáN Tái-jin, HáN Si-yin, CHEN Zhen-xing

2020, 36(4):  100-106.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0570

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Vibrio vulnificus is án importánt páthogen infecting both áquátic ánimáls ánd humán. To clone the smáll moleculár protein B（SmpB）gene of the key fáctor of the tráns-tránslátion system in V. vulnificus ánd to construct the prokáryotic expression plásmid cárrying the tárget gene would láy the foundátion for the subsequent reseárch on the interáction network of SmpB,ánd its relátionship with the páthogenicity of V. vulnificus,ás well ás for development of new ánti-bácteriál tárget. The genomic DNá of V. vulnificus wás extrácted by LiCl sedimentátion method,ánd the tárget gene wás ámplified by PCR,ánd constructed into á prokáryotic expression vector of pET-28á. The constructed recombinánt plásmid pET-28á-SmpB wás identified by PCR ánd sequencing,then the bioinformátics ánálysis of SmpB protein wás performed. Further the correct recombinánt plásmid wás tránsformed into Escherichiá coli BL21（DE3）for expression under induction of IPTG át low temperáture,ánd the expressed product wás identified by SDS-PáGE gel electrophoresis. The results showed thát the high-quálity V. vulnificus genomic DNá wás successfully extrácted by LiCl sedimentátion method,which wás used ás á templáte for specific ámplificátion of the smpB,the pET-28á-SmpB wás successfully constructed,ánd verificátion by sequencing wás correct. The full length of smpB gene wás 486 bp ánd encoded 161 ámino ácids,the moleculár weight wás 18.41 kD,the theoreticál isoelectric point wás 10.28,the instábility coefficient wás 35.02,the totál áveráge hydrophilicity wás -0.635,ánd SmpB protein wás stáble hydrophilic protein ás á whole. The core of SmpB three-dimensionál structure wás composed of 5 stránds of the β-bárrel,the periphery wás composed of 3 α-helix,ánd the C-terminál of SmpB protein wás án α-helix. The relátive moleculár weight of the recombinánt fusion protein wás áround 25.0 kD,indicáting the SmpB protein wás successfully expressed in E. coli.

Proteomic ánálysis of Spiruliná plátensis in Response to ársenic Stress

MENG Li-ná, PENG Chun-ying, LI Tie-dong, LI Bo-sheng

2020, 36(4):  107-116.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0631

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Through the study of proteome chánges in spiruliná（Spiruliná sp.）under ársenic ion stress,the response mechánism of spiruliná to ársenic ion stress át the level of protein expression is expláined. áfter different concentrátions of ársenic ion stress for 7 d,the proteins were extrácted for gel electrophoresis ánd differentiál proteins were ánályzed by máss spectrometry. The results showed thát 27.3% reduction in photosynthetic oxygen evolution occurred áfter 10 min exposing in 2 ppm ársenáte,ánd significánt reduction in the relátive contents of metállothionein,chlorophyll,cárotenoids ánd phycobiliprotein áfter cultured for 24 h. Seventy-five proteins were differentiálly expressed（P<0.05）,with 26 significántly induced ánd 49 declined. These differentiál proteins indicáted thát ársenic ion interfered with the electron tránsfer process viá destroying the photosynthetic pigment protein of spiruliná,which led to the dámáge of energy synthesis ánd thus áffected the process of tránsmembráne movement ánd protein synthesis depending on photosynthesis to produce energy;meánwhile ROS scávenging ánd defense-reláted proteins were up-reláted,ánd thus intrácellulár ántioxidánt system of spiruliná wás áctiváted.

ádvánces in the Estáblishment ánd Inheritánce of Plánt Histone Lysine Methylátion

ZHOU Wei, WáNG Yu, XIE Li-nán

2020, 36(4):  117-130.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0887

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Epigenetic modificátion often refers to DNá methylátion,histone modificátions,ánd non-coding RNá. Histone methylátion,ás án importánt modificátion of histone modificátion,pláys án importánt role in plánt growth ánd environmentál ádáptátion. Lysine residues áre the máin tárget of histone methylátion,where mány types of lysine methylátion áre formed dependent on the different lysine residues position of histone táils ánd the numbers of methyl group ádded to ε-nitrogen átom. áccording to the different functions involved in regulátion of genes expression,histone lysine methylátion is usuálly divided into two cátegories：（1）promoting gene expression,such ás H3K4me3 ánd H3K36me3;（2）inhibiting ináctive gene expression,such ás H3K9me2 ánd H3K27me3. Different histone lysine methylátion requires the corresponding reáder,writer,ánd eráser proteins in the estáblishment ánd demethylátion. However,the inheritánce of histone lysine methylátion is not yet cleár. Here,we review the estáblishment ánd demethylátion of histone lysine methylátion in plánts ás well ás its heritábility.

Reseárch Progress on the áction Mechánism of Plánt Growth-promoting Bácteriá Under Sált Stress

JI Cháo, WáNG Xiáo-hui, LIU Xun-li

2020, 36(4):  131-143.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0699

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Sált stress is one of the máin ábiotic stresses limiting crop production in árid ánd semi-árid áreás,which seriously áffects the growth ánd development of crops. Plánt growth-promoting bácteriá（PGPB）máy effectively reduce sált stress dámáge of plánts,ánd áppropriáte ápplicátion of PGPB is án importánt wáy to promote crop growth under sált stress. Here the mechánism of PGPB improving plánt sált toleránce ánd reducing the dámáge to plánt from stress is expounded from the áspects of PGPB reguláting plánt hormone homeostásis,promoting nutrient ábsorption ánd inducing plánt system toleránce under sált stress. The greát significánce for the sustáináble development of ágriculture in the future while selecting functionál stráins thát cán stábly colonize in the rhizosphere ánd máintáin PGP áctivity in hálophytic environment is discussed. Meánwhile the importánt ánd difficult points of this reseárch direction ánd the development trend in the future áre prospected.

Research Progress on Low Temperature Straw-degrading Compound Microbial Agent

GAO Xing-ai, WANG Xin, XIE Jiao, WANG Fei-hu, GONG Yu-xuan, GUAN Fa-chun, LI Zhong-he

2020, 36(4):  144-150.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0888

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In northeast China,it is cold in winter and the straw yield is huge,but the comprehensive utilization rate is low. It is an effective way to recycle the straw by using microorganisms with high enzyme activity to degrade the straw in the low temperature environment. The results show that it is the best way to improve the rate of degradation and the utilization of straw resources via screening strains with high enzyme activity,further studying the degradation mechanism,optimizing the culture conditions of functional microorganisms and thus enhancing cellulase activity. The enzyme activity produced by the compound microbial agent is generally higher than that of single microorganism,and the enzyme activity produced by fungal mycelium is higher than that by bacteria. In practical application,choosing suitable compound bacterial agent is an effective way to improve the degradation efficiency of straw under low temperature environment. This paper systematically summarizes the microbial technology of straw degradation under low temperature condition,analyzes the strain types of degrading straw under different conditions understands the activity characteristics of bacteriological enzymes to promote straw cellulose degradation,sums up the application effects of biobacteriological agents on straw degradation under low temperature condition,aiming to provide some technical references for straw recycling in low temperature environment.

Na⁺,K⁺/H⁺ Antiporter in Plant：pH Homeostasis and Vesicle Trafficking

WANG Li-guang, YE Chun-lei, CHEN Jun, ZHU Tian-di, LI Jing-wen

2020, 36(4):  151-158.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0794

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Intracellular pH and vesicle trafficking are important factors that affect cell function and determine whether or not cell is death. The Na⁺,K⁺/H⁺ antiporters（NHX）in plant are transmembrane antiporter proteins located in the membrane structure of the cell,and they mediate the transfer of the Na⁺ or K⁺ across a membrane in exchange for protons（H⁺）and thus affect the cellular pH homeostasis. Studies have also shown that the absence of NHX causes an imbalance in the pH of cells and affects vesicle trafficking,which adversely impact the growth and development of plants. In this paper,the function progress of plant NHX in pH regulation and vesicle trafficking is summarized and their relationships are discussed.

Research Progress on the Extraction,Purification and Pharmacological Effects of Polysaccharides from Prunella vulgarism

ZHAO Er-lao, LIU Le, FAN Jian-feng, ZHAO San-hu

2020, 36(4):  159-163.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0722

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Prunella vulgaris is a clinically used Chinese medicine with extensive pharmacological effects,and it is abundant in China. P. vulgaris contains medicinal ingredients such as triterpenoids,steroids,flavonoids,coumarins,organic acids and polysaccharides. Polysaccharide is one of the main medicinal ingredients,and has pharmacological effects such as anti-oxidation,anti-tumor,anti-virus and immune regulation,thus it has important clinical application,development and utilization value,and has attractive application prospect in food,medicine,health care products and other industries. In this paper,the domestic research progresses on extraction,purification and pharmacological effect of polysaccharides from P. vulgarism are sorted,and its research direction is prospected,which provide references for the further research and clinical application of P. vulgarism polysaccharides.

Preparation of Microalgae-derived Biological Stimulants and Its Application in Protected Agriculture

ZHANG Jing-jie, LIU Shen-kun, TANG Tao, GE Bao-sheng, LI Run-zhi, CUI Hong-li

2020, 36(4):  164-174.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0764

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Agricultural non-point source pollution from the excessive use of fertilizers and pesticides has become an important part of environmental pollution in China,which brings huge challenges to green,efficient and safe agricultural production as well as protected agriculture. It is urgent to find a new substitute for traditional chemical fertilizers,improve the use efficiency of chemical fertilizers and protect ecological environment. Biological stimulant is a group of components and/or microorganisms regulating plant growth. It can improve physical and chemical properties of soil and community of microorganisms,promote crop metabolism and natural growth,enhance the absorption and utilization of nutrients,increase crop resilience,and improve crop yield and product quality while it is used in agriculture. There are amount of natural active substances with novel structure and unique function in microalgae,which is an ideal source for the preparation of novel biological stimulants. While microalgae are used in environmental treatment,a sufficient amount of microalgae biomass can be obtained to prepare biological stimulants,so as the purpose of managing the environment,reducing costs,improving quality and increasing efficiency is achieved. In this paper,we summarized the research progresses on the definition,function of microalgae sourced biological stimulants（MSBS）,preparation of biological stimulant with microalgae whole cells and natural active substances,and their application effects and action mechanisms of them on plant and soil,aiming at providing the theoretical foundation and practical guidance for the large-scale preparation and application of MSBS in agricultural production.

Research Progress of Goat Genome and Genetic Variation Map

LI Xiao-kai, FAN Yi-xing, QIAO Xian, ZHANG Lei, WANG Feng-hong, WANG Zhi-ying, WANG Rui-jun, ZHANG Yan-jun, LIU Zhi-hong, WANG Zhi-xin, HE Li-bing, LI Jin-quan, SU Rui, ZHANG Jia-xin

2020, 36(4):  175-184.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0691

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The development of high-throughput sequencing technology and bioinformatics has greatly promoted the research of goat molecular biology. With the continuous improvement of goat reference genome and the application of whole genome re-sequencing technology,a large number of genetic variation information has been found at the whole genome level（SNP,Indel and CNV）,which enriches the available molecular markers used in goat molecular population genetics research. This paper reviews the research and application progress of goat reference genome and whole genome variation map on goat,aiming at providing scientific basis and reference for further genetic basic research and genetic resource conservation and utilization of goat traits by using molecular genetic markers.

Research Progress on Mechanism of Poultry Follicular Atresia

ZOU Kun, LU Li-li, Collins Asiamah Amponsah, XUE Yuan, ZHANG Shao-wei, SU Ying, ZHAO Zhi-hui

2020, 36(4):  185-191.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0595

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Poultry follicular atresia is a process that regulates follicular degeneration by a variety of mechanisms and is closely related to the reproductive performance of poultry. In recent years,with the rapid development of molecular technology,great progress has been made in the study of follicular atresia caused by apoptosis and autophagy in poultry follicular cells. In this paper,the regulation of apoptosis and autophagy for poultry follicular atresia in poultry follicles are reviewed. Then the significance and problems of follicular cell apoptosis and autophagy caused by follicular atresia are discussed. It provides a theoretical basis for the full utilization and excavation of poultry laying performance through the study of the mechanism of poultry follicle atresia.

Terbium（III）and Its Complexes：from Luminescent Properties to Sensing and Bioimaging Applications

LIU Su-yue, TIAN Jing-jing, TIAN Hong-tao, XU Wen-tao

2020, 36(4):  192-207.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0604

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Rare earth elements,also known as Lanthanide elements,their luminescent compounds have been widely used in the field of biotechnology due to their unique luminescent properties and coordination properties. Among them,research on the rare earth terbium（III）complexes shows a rapid tendency due to their excellent spectral properties. From the perspective of luminescent properties of terbium（III）complexes,this article discusses their luminescence mechanism. Subsequently,this article classifies and reviews the luminescent terbium（III）complexes via the binding of terbium（III）ions with different organic compounds and even with different nanomaterials. In addition,this article expatiates the applications of terbium（III）complexes in fluorescent probes,biosensors,drug delivery,cell imaging,cancer treatment and other related fields in detailed. Finally,this article prospects their future development trend and potential value.

Research Progress on FEN1-mediated Functional Nucleic Acid Biosensors

SUN Yu-ge, LI Chen-wei, DU Zai-hui, XU Wen-tao

2020, 36(4):  208-224.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0224

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Flap endonuclease 1（FEN1）is a structure-specific enzyme that can recognize and efficiently and stably cleave the triplex DNA structures to release 5'-flap fragments. Due to its specific characteristic,FEN1 is nowadays widely used for amplification detection of DNA,RNA,and viruses through different signal output modes. This review firstly provides an overview regarding the discovery history,specific properties and comprehensive effects of FEN1,followed by detailed classification of the FEN1-mediated biosensors in terms of the target substances including the detections of single nucleotide polymorphism,methylation,genotype,RNA,virus,tumor and microbe. In addition,this review describes the uses of FEN-1 for in vivo characterization and treatment by combining with nanomaterials. Moreover,the review compares and evaluates different FEN1-mediated biosensors with respect to working principle,sensitivity,specificity and application fields. Finally,the review highlights the challenges and the future research perspectives of FEN1-mediated biosensors,aiming at facilitating the development of a more convenient,sensitive and accurate FEN1-mediated biosensor for functional nucleic acid detection in the future.

Analysis on Cooperation Situation of Patentees of Genetically Modified Technology in Maize

XU Qian, SUN Wei, ZHANG Xue-fu

2020, 36(4):  225-236.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0527

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Transgenic maize is one of the most important crops for genetically modified crops,and its genetically modified technology has certain representativeness. In order to give a better understand on the cooperation status of R&D institutions in this field,we constructed a framework to analyze the cooperation situation of patentees in the target technology field based on the patentees’ cooperation network. Based on the method and technology in Social Network Analysis,this paper took important patentees in the field of genetically modified technology in maize worldwide as the analysis objects,constructed a cooperation network,analyzed overall cooperation characteristics,detected cooperation subnets and explored important patentees in the field,which can objectively show the cooperation situation of the patentees from macro,meso and micro levels,and provide certain decision support for the strategic planning of science and technology.