Biotechnology Bulletin

Research Progress for the Gibberellin Signaling and Action on Plant Growth and Development

GAO Xiu-hua, FU Xiang-dong

2018, 34(7): 1-13. doi:10.13560/j.cnki.biotech.bull.1985.2018-0447

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Gibberellins（GAs）are the important plant hormones and play an important role in modulating diverse processes throughout plant growth and development. Recent studies on GA signaling and the crosstalk between GA and other plant hormones and environmental cues have achieved great progress along with the advancement of molecular genetics and functional genomics. Bioactive GAs promote plant growth and development by promoting the degradation of the DELLA proteins,a family of nuclear growth repressors. In this article,we review the key components of GA signaling pathway,including the soluble receptor protein GIBBERELLIN INSENSITIVE DWARF1（GID1）,the F-box protein（SLEEPY1[SLY1]in Arabidopsis and GIBBERELLIN INSENSITIVE DWARF2[GID2]in rice）and the DELLA proteins,to demonstrate the GA's “de-repression” model,we also focus on DELLA proteins function which is connected to developmental and environmental responses.

Jasmonate Action and Biotic Stress Response in Plants

WU De-wei, WANG Jiao-jiao, XIE Dao-xin

2018, 34(7): 14-23. doi:10.13560/j.cnki.biotech.bull.1985.2018-0442

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Being sessile,plants are always challenged by a variety of environmental stresses,including herbivorous insect attack and microbial pathogen infection. To defend themselves against these stresses,plants have evolved sophisticated and highly regulated defense system,such as activation of biosynthesis of plant hormones that regulate defense-related gene expression and secondary metabolite production. Intriguingly,many herbivorous insects and microbial pathogens have been found to employ distinct strategies to evade,overcome,or even manipulate plants' defense system to facilitate their exploitation of host plants. Jasmonate,an important class of lipid-derived plant hormone,regulates multiple aspects of plant growth and development and also plays crucial roles in plant responses to diverse biotic and abiotic stresses. In recent years,the biosynthesis,signal transduction and biological functions of jasmonate have been extensively studied,and significant progresses have been achieved. In this review,we provide a overview of jasmonate biosynthesis regulation and signaling transduction pathway. We also discuss the mechanisms of jasmonate action in plant biotic stress responses and summarize the various strategies used by herbivorous insects or microbial pathogens to hijack jasmonate pathway. This review would be helpful for an in-depth understanding of jasmonate-mediated interactions of plants with pathogenic organisms.

Research Advances of Auxin Signal Transduction

FENG Han-qian, LI Chao

2018, 34(7): 24-30. doi:10.13560/j.cnki.biotech.bull.1985.2018-0488

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The phytohormone auxin plays a crucial role in almost every aspect of plant growth and development. The function of auxin ranges from cell expansion,cell division,to cell differentiation,as well as the biological processes from embryogenesis to reproduction. The auxin research is focused on three aspects：auxin synthesis and metabolism,auxin polar transport,and auxin signal transduction. Auxin response is mainly mediated by the nuclear TIR1/AFB-Aux/IAA-ARF signaling network. Besides,some fast auxin responses are regulated by a supposed cell surface initiated auxin signaling pathway. A SKP2A pathway has been identified to regulate cell cycle. In this review,we summarized the progress on auxin signal transduction pathways,as well as introduced the research tools for the auxin signaling transduction studies. We proposed the future directions for auxin signal transduction studies,including the regulation of TIR1/AFB-Aux/IAA-ARF complex,the differential auxin modulation in shoot and root,and the auxin signaling on the plasma membrane.

Mechanism of Plant Development Regulation by Endomembrane Trafficking

PANG Lei, ZHU Ying, JIN Zhong-cai, XIA Xi-hua, LI Rui-xi

2018, 34(7): 31-39. doi:10.13560/j.cnki.biotech.bull.1985.2018-0360

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Similar to other eukaryotic system,plant endomembrane system is a functionally inter-related membrane system including the nuclear envelope,the endoplasmic reticulum（ER）,the Golgi apparatus,the trans-Golgi network（TGN）,vacuoles,endosomes,plasma membrane（PM）and vesicles. It also displays some highly complex and unique characteristics,such as the unique feature of plant TGN and vacuole. This review mainly summarizes the regulatory mechanisms of plant endomembrane trafficking in plant development. We focus on the recent progresses about how endomembrane system regulate auxin polar transport and homeostasis,mechanism of root hair tip growth as well as the vacuole transport in the regulation of pollen tube tip growth and seed germination. We hope this review could be helpful to the researchers who study the process of plant growth,development and endomembrane trafficking.

Regulatory Mechanism of Mediator Subunit MED25 on Multi-phytohormone Signaling Pathways

DOU Yue, LIU Mei-tong, LU An-na, WU Jia-jie, WANG Qun-qing, XU Qian

2018, 34(7): 40-47. doi:10.13560/j.cnki.biotech.bull.1985.2018-0487

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The multi-subunit mediators regulate gene expression by bridging RNA polymerase II and kinds of transcription factors into Pre-Initiation Complex to trigger specific transcriptional program. Mediator25（MED25）is one subunit of the plant mediator complex,implicated in regulating plant development and stress tolerance. MED25 participated in response of biotic and abiotic stresses by interacting with transcription activators（e.g.,MYCs,AP2/ERFs and ARFs）,repressors（e.g.,JAZs and Aux/IAAs）and other mediator subunits（e.g.,MED13 and MED16）,and regulation of phytohormone（JA,ABA,ethylene and IAA）signaling pathways. The target separation and functional identification of MED25 have been a hot topic in recent years. In this paper,the essential roles of MED25 in various hormone signaling pathways were reviewed and provide novel insights into how the plants pass hormone-specific regulatory signals and transcriptionally control physiological processes.

Research Progress on the Effects of Phytohormones on Crop Root System Development Under Drought Condition

GUO Bin-hui, DAI Yi, SONG Li

2018, 34(7): 48-56. doi:10.13560/j.cnki.biotech.bull.1985.2018-0425

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Phytohormone is a kind of trace organic that are synthesized in parts of plants and can be transported to other functional parts of plants to regulate plant growth and development. As an important organ for crops to absorb water and nutrients, root system development determines the ability of crops to obtain nutrients and water. Recent studies indicate that the well-developed root system of crops is closely related to their resistance to drought stress and phytohormones play a key role in the root system development. Therefore, it is important to understand the effects of plant hormones on crop root development under drought stress. In this paper, we summarized recent evidences on the roles of phytohormones independently or synergistically affecting the crops root system development under drought condition, and discussed the significance of applying phytohormones in crop drought resistance research and the feasible research directions in future.

PIF4-Regulated Thermo-responsive Genes in Arabidopsis

WANG Shuo, DING Lan, LIU Jian-xiang, HAN Jia-jia

2018, 34(7): 57-65. doi:10.13560/j.cnki.biotech.bull.1985.2018-0211

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Plant growth and development programs are coordinated with various internal factors such as phytohormones and environmental signals such as fluctuated temperatures. The Phytochrome B（PHYB）interacting protein PIF4 promotes plant morphogenesis at elevated ambient temperature,however,genome-wide downstream genes of PIF4 during this response have not been reported. In this study,we confirmed the important role of PIF4 in thermoresponsive morphogenesis in Arabidopsis,and 248 PIF4-dependent and 21 PIF4 partially dependent downstream genes at elevated ambient temperature were further revealed by RNA-Seq,including those genes in auxin responsive pathway. 74 possible direct targets of PIF4 in thermomorphogenesis were identified by combining the previous published CHIP-Seq data and our RNA-Seq data,and 6 out of 7 were confirmed by ChIP-qPCR. Thus,our study has revealed PIF4 downstream genes during thermomorphogenesis,which are important for a better understanding of plant responses to elevated ambient temperature.

Effects of GA and B9 on the Growth and Development of Potato Tubers

HE Yi-xue, LIU Wen, SHEN Xiang-ling

2018, 34(7): 66-73. doi:10.13560/j.cnki.biotech.bull.1985.2018-0023

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Potato is the fourth largest food crop in the world and thus possesses high economic value. Potato tubers are an important part of the research on potato breeding and reproduction,which is of significance on the research and application of potato. In order to explore the effects of gibberellin（GA）and butyryl hydrazine（B9）on the growth and development of potato tubers,different concentrations of GA and B9 were used for orthogonal experiment. Based on the statistics results of potato plant height,lateral shoots,leaves,tubers' number,and tuber starch content,the effects of adding both GA and B9 on the growth and development were significant,and especially significantly greater than that of adding GA or B9 alone on the number and quality of potato tubers. Moreover,the addition of 0.5 mg/L GA and 20-40 mg/L B9 medium contributed to the formation of potatoes with large leaves,stalks,tuber number and tuber starch content.

Effect of Sucrose,GA and Liquid Medium on the Tuber Formation of Potato

HE Yi-xue, LIU Wen, SHEN Xiang-ling

2018, 34(7): 74-80. doi:10.13560/j.cnki.biotech.bull.1985.2018-0355

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Potato is the fourth largest food crop in the world. It has been used for both grain and vegetable and is rich in nutritional value. Potato tubers are the most economically valuable part of the potato. The formation process of potato tuber is regulated by a variety of regulatory pathways,the most important ones of which is the sucrose pathway and the gibberellin（GA）pathway. This experiment was designed to conduct orthogonal testing three factors of sucrose concentration,GA concentration and media type. By the statistics of potato tuber formation time,the number of potato tubers,potato average weight,and the rate of large potato,it was found that high concentration of sucrose advanced potato tubering,and liquid medium increased the potato weight and the rate of large potato. The addition of 8% sucrose and 0.1 mg/L GA in liquid MS medium resulted in advanced potato tuber formation as well as more and heavier potatoes.

Effects of Methyl Jasmonate on Active Ingredient Content in Hairy Roots of Salvia miltiorrhiza

XIONG Bing-quan, LIU Dong-qing, LIAO Xiang-jian, ZHENG Xue-lian

2018, 34(7): 81-84. doi:10.13560/j.cnki.biotech.bull.1985.2017-1001

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The effects of methyl jasmonate（MJ）on the content of secondary metabolites in Salvia miltiorrhiza hairy roots were studied to provide experimental basis for the production and application of culturing hairy roots of S. miltiorrhiza. Having methyl jasmonate as the inducer,the hairy root culture of S. miltiorrhiza was induced. The results showed that the contents of tanshinone Ⅰ and tanshinone ⅡA of S. miltiorrhiza hairy roots with MJ were 1.53 times and 1.16 times higher than those of hairy roots without MJ respectively. Similarly,the contents of rosmarinic acid and salvianolic acid B were 1.37-fold and 4.43-fold increase in S. miltiorrhiza hairy roots with MJ. The results confirmed that MJ was an efficient inducer,which significantly improved the synthesis and accumulation of the secondary metabolites in S. miltiorrhiza hairy roots. In another words,induction by MJ could be used for the efficient production of effective secondary metabolites of S. miltiorrhiza.

Expression Analysis of Mitochondria-related Genes During PCD Induced by Salicylic Acid and Downy Mildew in Cucumber

WANG Dan-dan, CHI Chun-ning, BAI Jing, CHEN Chong, CHI Chun-yu, DING Guo-hua

2018, 34(7): 85-91. doi:10.13560/j.cnki.biotech.bull.1985.2018-0471

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In order to study the differences in the expression of mitochondria-related genes during the process of cucumber PCD induced by salicylic acid（SA）and downy mildew as well as the way of PCD occurred in cucumber leaves,the leaves were treated with 10 mmol/L SA and inoculated with the spore suspensions（5×10⁵-10×10⁵Spores /ml）of Pseudoperonospora cubensis at the four-leaf period of cucumber. Total RNAs were extracted from the leaves treated with SA after 0,3,12,and 24 h and the leaves treated with P. cubensis after 0,36,72,and 96 h. The expressions of the 16 mitochondria-related genes were analyzed using qRT-PCR techniques. The results showed that the 16 genes expressed after SA-treatment and P. cubensis infection with a similar or contrary expression trend,indicating that the genes with PCD induced by SA were significantly related to the signaling pathway of PCD induced by downy mildew. Eleven genes of them were up-regulated both in downy mildew treatment and in SA treatment,5 genes were up-regulated in SA treatment,but were down-regulated in downy mildew treatment,showing that there were differences in disease resistance signaling pathways induced between by SA and downy mildew,which then resulted in the different occurrence patterns of PCD in cucumber leaves. These 16 genes belonged to 4 functional groups,including mitochondrial membrane transport,cell respiration,protein synthesis,folding and transport,mitochondrial signal transduction and other unknown functions.

Agronomic Traits Analysis of Transgenic Bt cry1Ah Maize HGK60 Line

LIANG Hai-sheng, LI Meng-tao, LI Sheng-yan, WANG Hai, ZHANG Jie, LANG Zhi-hong

2018, 34(7): 92-100. doi:10.13560/j.cnki.biotech.bull.1985.2018-0219

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To determine the genetic expression stability and agronomic traits of the inbred lines of Bt cry1Ah-resistant maize HGK60 and its hybrid progenies,we analyzed the genetic expression stability of the exogenous genes by fluorescence quantitative PCR and ELISA,both bioassay indoor and outdoor. Analysis of agronomic traits were carried out by bioassay and agronomic traits considerations. The result of fluorescent quantitative RT-PCR showed that Bt cry1Ah could be transcribed normally in different plant tissues,and the expression level of RNA was different. The results of ELISA showed that the protein expression of Cry1Ah in different organs at different developmental stages：tassel > leaf > husk > grain > filament > cob. Through two locations and three consecutive generations of bioassay indoor and outdoor,the HGK60 line showed high resistant to Asian corn borer. Compared with the recipient inbred and hybrid lines,seed germination rate,growth period,ear number,spike length,thousand grains weight and other agronomic traits considerations indicated are not significantly different. Through experiments and molecular tests tracking over many years,the genetic and expression stability of the exogenous genes in HGK60 transgenic maize has been proven to be very resistant to Asian corn borer,and there is no significant difference between agronomic traits and control materials. The good resistance of HGK60 transgenic insect-resistant corn to Asian corn borer proved that HGK60 transgenic insect-resistant maize has good commercial application prospect.

Cloning and Functional Analysis of the MtLEA5B Gene from Medicago truncatula

ZHANG Ye-meng, SHEN Ying-fang, WANG Ying-fang, YAO Pin-ya, WANG Hai-qing

2018, 34(7): 101-107. doi:10.13560/j.cnki.biotech.bull.1985.2018-0095

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Late embryogenesis abundant protein（LEA protein）is hydrophilic one that is involved in the responses to stress in plants. This work is aimed to unravel the protective effect of heterologous expressed LEA protein on host under abiotic stress. A novel gene encoding LEA_5 protein was cloned from Medicago truncatula seedlings,and designated as MtLES5B,and its expression patterns were analyzed by semi-quantitative RT-PCR. Then the heat resistance and solubility of recombinant protein were detected by SDS-PAGE protein electrophoresis. Further,the prokaryotic expression vector was constructed and transferred to Escherichia coli so as to induce MtLEA5B over expression in E. coli. The expression patterns of MtLEA5B gene was regulated by dehydration,low temperature,high salt stress,and ABA induction. There was a band by SDS-PAGE protein electrophoresis,which was larger than the theoretical prediction value and did not decrease in solubility under boiling water condition. The overexpression of MtLEA5B protein in E. coli obviously increased the survival rate of hosts at heat（55℃）and freeze（-20℃）. Moreover,MtLEA5B was an inducible expressed gene,MtLEA5B protein had high hydrophily and heat resistance,overexpression in E. coli may significantly enhance its tolerance to temperature stress.

Cloning and Expression of EsABCG25 from Eutrema salsugineum

Songbuerbatu CHEN Yue CHEN Ning-mei TANG Shuai XU Xiao-jing

2018, 34(7): 108-118. doi:10.13560/j.cnki.biotech.bull.1985.2018-0020

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This work is aimed to reveal structure of ABCG25 and explore its response to drought,salinity and temperature stresses in Eutrema salsugineum. A full-length cDNA of member gene in ATP-binding cassette transporters（ABCG）family was cloned from E. salsugineum（Shandong ecotype）using silicon cloning and RT-PCR methods,and named as EsABCG25 with GenBank accession number of KY111263. The full length of EsABCG25 was 2154 bp and it contained a complete ORF with 1983 bp and encoded 660 amino acids. EsABCG25 was located at chromosome 5,with 4 exons and 3 introns. EsABCG25 had a putative nucleotide binding domain（NMD）and a putative transmembrane domain（TMD）. Alignment and phylogenetic analysis found that EsABCG25 was the closest to the ABCG25 from Brassica rapa. High-throughput sequencing indicated that EsABCG25 reached the highest expression in the stem of E. salsugineum and the lowest expression in rosette leaves,and alternative splicing events were detected in all tissues. Real-time PCR assay indicated that EsABCG25 was induced by many kinds of stresses,especially ABA stress. From above,EsABCG25 has typical characteristics of ABCG gene family. Its expression variedat different tissues,was induced by stress,and may be closely associated with the stress tolerance of E. salsugineum.

Development and Phenotypic Analysis of Tetraploid Arabidopsis thaliana with QUARTET Mutation

HE Shuo-kang, LUO Ze-wei

2018, 34(7): 119-125. doi:10.13560/j.cnki.biotech.bull.1985.2018-0238

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To apply tetrad analysis in the research on genetic recombination of polyploid,we treated the diploid Columbia（Col）from gene qrt1 mutation and Arabidopsis thaliana of Landsberg erecta（Ler）ecotype with colchicine for inducing the genome doubling. The flow cytometry and fluorescence in situ hybridization were employed to screen the successfully-doubled tetraploid Col and Ler from gene qrt1 mutation. Then their vegetable and reproductive phenotypes were analyzed. As results,firstly,qrt1 mutation reduced the number of lateral roots and side bolts and the area of rosette leaves. Secondly,the area of rosette leaves and the size of pollen,anther and seed of the tetraploid increased,and the number of lateral roots and side bolts decreased,and the time of bolting siliques delayed,And the number of fruit pods per plant and seeds per fruit pod were less,while the germination rate approached 100%. Thirdly,some phenotypes were influenced differently by ploidy in Col and Ler,for example,the effects of ploidy on the height of stem,the number of branches and length of siliques varied.

Revealing Hybridization of Brassicaceae Based on Comparative Genomics

FAN Xiao-meng, QI Ji

2018, 34(7): 126-137. doi:10.13560/j.cnki.biotech.bull.1985.2018-0338

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Hybridization is one of the sources for the evolutionary dynamics of species. It helps to rapidly accumulate genetic variation,to enhance the genetic diversity of the population,and to promote the production of new species. Therefore,it is of great significance to identify ancient and recent crosses in different species groups. Identification methods of hybridization event may employ a variety of features,including traditional genetic phenotypes,metabolites,chromosome numbers,and molecular phenotypes of gene levels provided by population genetics and phylogenetic genomics. In this study,28 Brassicaceae species and two species of other family were selected as research materials,and comparative genomics and phylogenetic genomics in the chloroplast and nuclear genes in combination with molecular phenotypic information were used to identify the Brassicaceae hybrid events. The results show that：1. Based on the results of mapping single species trees to species trees,Brassicaceae species are mainly classified into two groups：one is that a sister group is relatively concentrated and represented by Arabidopsis thaliana;another is that a sister group is relatively scattered,suggesting there are multiple sources of genes,represented by Lunaria annua. 2. Further,by phylogenetic analysis of chloroplast and nuclear genes,we believe that L. annua might be a new hybrid species,39.1% of its genes are most closely related to the parental C group genes;38.5% of the genes are the closest to the genes of non C groups. In particular,the No. 10 node from the common ancestors of the B and C binding groups（accounting for 3%）supports itself as a maternal source of L. annua. 3. By the functional enrichment analysis of the key evolutionary nodes of the parents,we find that the male parent is mainly enriched in biodegradation,secondary metabolites and RNA regulation,indicating that it may regulate biological degradation and secondary metabolic processes to adapt to the stress environment via RNA;maternal enriched in the metabolic pathways,especially carbohydrates,nucleic acid metabolism,etc. indicating that it may obtain relative competitive advantage by enhancing its own metabolism.

Purity Identification and Genetic Diversity Analysis of Cotton Germplasm Resources Using SSR Markers

SHI Jian-bin, ZHOU Hong, WANG Ning, XU Qing-hua, QIAO Wen-qing, YAN Gen-tu

2018, 34(7): 138-146. doi:10.13560/j.cnki.biotech.bull.1985.2017-1070

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The purity and genetic diversity of 58 cotton germplasm resources were analyzed with 26 core SSR polymorphic markers screened in previous studies. The results showed that the purity of different materials was between 20%-90%,and the detection result of molecular marker was conformity with the phenotype in field,indicating that the accuracy of SSR detection was high and it can be used as a method for rapid detecting the purity of cotton germplasm. A total of 85 polymorphic genotypes were obtained,and the number of genotypes detected by each marker was between 2 and 5,with an average of 3.27. The primer polymorphism information content was between 0.073 7-0.928 1,with an average of 0.363 9. NTSYS-pc2.10 software was used to analyze the genetic diversity of 58 cotton germplasm resources,the results showed that the genetic similarity coefficient ranged from 0.305 1 to 0.898 3,the amplitude of variation was 0.593 2. The 58 materials can be divided into 5 major categories based on 0.63 GS level,and the variety resources of DNA clustering showed no correlation with their geographic ecological source,but a high correlation with genetic relationships of the materials. The clustering result can more truly reflect the genetic differences between the varieties.

Isolation and Similarity Analysis of Epiphytic and Endophytic Bacteria in Different Tissues of Tomato Plants

WANG Ling-ling, SHANG Qing-mao, DONG Chun-juan

2018, 34(7): 147-153. doi:10.13560/j.cnki.biotech.bull.1985.2018-0118

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In order to explore the correlation among environmental,epiphytic and endophytic bacteria associated with tomato plants,tomato cultivar ‘Zhongza 302' was used as experimental material,and traditional plate culture method,16S rRNA copies analysis,and denaturing gradient gel electrophoresis（DGGE）assay were applied to clarify the quantity distribution and community similarities of the bacteria isolated from root zone soils and rhizosphere,as well as the epiphytic and endophytic bacteria in various tissues,including roots,stems,leaves,fruits and seeds. The results showed that the number of rhizospheric bacteria was the highest,followed by the bacteria in root zone soil,and the epiphytic bacteria were much less than those from root zone soils and rhizosphere samples. Among different tissues,the number of root epiphyte was the highest,and then stem and leaf were followed. The numbers of endophytic bacteria were 2-4 orders of magnitude lower than those of epiphyte. Among different tissues,the endophyte from root was the most,while that from seed jelly was the least. DGGE analysis showed that the similarities between rhizosphere and other samples were higher compared to the root zone soils. The highest similarity was detected among endophytic samples from different tissues;especially for the adjacent tissues of roots,stems,and leaves,as well as seeds,placenta,and seed jelly,the similarities were above 0.6. These results indicate that the endophytic bacteria communities in different tomato tissues are very similar,and they are closely related to the rhizosphere bacteria.

Preparation and Identification of Polyclonal Antibody Against Calponin from Helicoverpa armigera

Nijiati·DILIMULATI, ZHAO Jie, AI Xin-yu, LIU Xiao-ning

2018, 34(7): 154-159. doi:10.13560/j.cnki.biotech.bull.1985.2017-1115

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Polyclonal antibodies against calponin from Helicoverpa armigera（HaCal）were prepared by protein immunization and used for preliminary application. The HaCal was expressed by the prokaryotic expression system,purified by Ni-column,and then immunized the mice by footpad and subcutaneous injection. The titer of polyclonal antibody against HaCal protein was determined by ELISA after four immunizations. The immunological specificity of the polyclonal antibody was detected by Western blot and immunohistochemical staining. The titer of mouse anti-HaCal polyclonal antibody prepared by protein immunization was higher than 1：819 200. Western blot and immunohistochemical staining showed that the antibody specifically recognized the native HaCal protein in the H. armigera. In summary,the polyclonal antibody against HaCal with high titer and specificity was prepared by protein immunization,which provided an important experimental tool for further studying the expression characteristics of HaCal protein and clarifying how to invovle in drug resistance of H. armigera.

Effects of Phoxim,Methomyl and Feeding dsRNA on the Expression of ace Genes in Helicoverpa armigera

ZHANG Ting, YANG Xuan-xuan, LEI Qin, LIU Juan-juan, LI Ji-gang

2018, 34(7): 160-165. doi:10.13560/j.cnki.biotech.bull.1985.2018-0004

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Acetylcholinesterase（AChE）is a target of organophosphorus- and carbamate-type pesticides. This study focuses on the acetylcholinesterase genes of Helicoverpa armigera,including the effects of phoxim,methomyl and feeding the ace dsRNA-expressing bacteria on the transcriptional activities of acetylcholinesterase genes,which may provide reference for the chemical control and RNAi-based control of H. armigera. The real-time quantitative PCR method was used to analyze the transcriptional levels of ace genes in phoxim- and methomyl-induced 4th instar larvae of H. armigera for 12 h,24 h,36 h,48 h,and 60 h. The 2th instar H. armigera larvae were fed with dsRNA-expressing bacteria,and the silencing effect of ace genes after RNAi was detected. Our results showed that the transcriptional levels of ace1 and ace2 were down-regulated at 12 h after phoxim and methomyl induction;subsequently,the transcriptional level of ace1 was down-regulated while the transcriptional level of ace2 was up-regulated. The transcriptional levels of ace1 and ace2 were down-regulated obviously at 24 h and 48 h then increased slightly after RNAi treatment. It is inferred that the pesticide treatment leads to the physiological and metabolic damages of H. armigera and consequently the significant reduction of transcriptional levels of ace1 and ace2;subsequently,the ace2 expression was decreasing constantly while the ace1 expression was gradually increasing,which might be related to compensation for pesticides' inhibition to AchE enzyme activity. RNAi treatment caused varied inhibitions to both growth and development of H. armigera,as well as the obvious inhibitions to the transcriptions of ace1 and ace2 genes. The above results provide a reference for the control of H. armigera while using acetylcholinesterase and its coding genes as the target.

Preparation and Regeneration of Protoplast from Verticillium dahliae

ZHAO Xiao-qiang, CHEN Zhi-rong, HE Fang, SHEN Nan, GAO Feng, HUANG Jia-feng

2018, 34(7): 166-173. doi:10.13560/j.cnki.biotech.bull.1985.2018-0160

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In order to establish optimal conditions on preparation and regeneration of protoplast from Verticillium dahlia,the single-factor experiment was used to analyze the effects of culture time,concentration of lyase,enzymatic digesting time and temperature,type and concentration of osmotic stabilizer,and pH on the release of protoplasts. The conditions of regeneration medium,agar concentration,and enzymatic lysing time on protoplast regeneration were optimized. In order to observe protoplast visually,the recombinant plasmid containing GFP was transformed by PEG-CaCl₂-mediated method. The results showed that the yield of protoplast was the highest with 3.3×10⁷cells/mL under the optimal conditions：collecting the mycelia when conidia cultured for 18 h,using 1.2 mol/L KCl as osmotic stabilizer,then adding 10 mg/mL lyase at pH 6.0,and lysing for 4 h at 30℃. Protoplast regeneration was achieved in TB3 medium with 0.5% agar,and the regeneration rate was up to 22.45%. The result from GFP transient-expression indicated that the protoplasts under optimal conditions may be used for genetic transformation.

Enhancement of Glucose Oxidase in Pichia pastoris by Co-expressing Chaperone PDI and Ero1

GAO Qing-hua, DONG Cong, WANG Yue, HU Mei-rong, WANG Qing-qing, WANG Yun-peng, LUO Tong-yang, LIU Lei

2018, 34(7): 174-179. doi:10.13560/j.cnki.biotech.bull.1985.2017-1095

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The expression of recombinant glucose oxidase（GOD）in Pichia pastoris was enhanced by the sole expression of protein chaperones protein disulfide isomerase（PDI）as well as the co-expression of endoplasmic reticulum oxidoreductase 1（Ero1）and PDI. The pPICZ/PDI and pPICZ/PDI -Ero1expression plasmids were linearized and integrated into the genome of P. pastoris X33/pMD-GOD by electroporation. The positive transformants were screened with double-resistance plate of 250 μg/mL G418 and 50 μg/mL Zeocin. The positive transformants were fermented in tube and 10 L fermenter for analyzing the effects of co-expressing PDI and Ero1-PDI on the expression of GOD. The results revealed that co-expression of PDI and Ero1-PDI increased GOD activity by 29.7% and 100% and their activity reached 476 U/m L and 736 U/m L respectively in 10 L fermenter at 30℃,compared with the control type of original strain. Conclusively,the integration of chaperone PDI and Ero1 enhanced the correct folding of GOD and significantly increased the expression of GOD.

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2018, 34(7): 200.

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