Peptides secreted by pathogens serve as elicitors of plant defense responses. Verticillium dahliae Aspf2-like protein (VDAL) is secretory protein of V. dahliae and belongs to Aspergillus fumigatus f2-like zinc-binding protein. The VDAL146 protein in V. dahliae Vd146 strain of cotton (Gossypium spp.) was made into dry powder by engineering bacteria fermentation, and its diluent was sprayed on the leaves, which could activate the immune response of plants such as rice (Oryza sativa) and promote the growth and development of plants. In present study, VDAL146 (MT974503) was isolated and characterized, and sprayed twice before and after rice break stage. The results showed enhanced resistance to rice sheath blight, as well as improved yield by 14.5% via increasing both the number of grains per panicle and thousand grain weight. At the same time, VDAL146 did not show direct fungicidal activity. In addition, transcriptome analysis indicated that VDAL146 treatment resulted in upregulation of 322 genes involved in carbohydrate metabolism, organophosphate and glucan biosynthesis, and ATP binding. In conclusion, VDAL146 holds the potential to be developed in fungicide and biostimulant to enhance disease resistance and growth promotion in rice crop.

Leaf color mutants are ideal genetic resources to reveal the mechanisms underlying chlorophyll biosynthesis. Here, a yellow leaf mutant yl(t), identified from indica rice (Oryza sativa) T98B was analyzed on phenotype, physiology characteristics and mutation mechanism. The results showed that, the seedlings of yl(t) presented yellow leaves with a significant decrease of 53.02%, 55.35% and 46.88% in the contents of total chlorophyll, chlorophyll a and chlorophyll b, respectively, compared with T98B. When it grew to mature, the chlorophyll content increased. Further genetic analysis showed yl(t) was controlled by a nuclear recessive gene. By employing a F₂ population from yl(t) crossed with the O. sativa subsp. japonica cv. Nipponbare (NPB) which presents normally green leaf, yl(t) was fine mapped in a 42 kb region on chromosome 6. Fragment sequencing confirmed that the heme oxygenase 1 gene OsHO1 (LOC_Os06g40080.1) would be the candidate of yl(t) that happened to mutate at the splicing site of exon2/intron2 (AG/GT) with a deletion of 'T'. Moreover, this point was proved to be associated with variation in leaf color by a CAPS marker's verification. Later, by means of 3' rapid amplification of cDNA ends (RACE), the full-length CDS of OsHO1 in yl(t) was cloned. Compared with T98B, the transcription of OsHO1 in yl(t) terminates at the 20~22 bp (TAA) on the second intron, thereby causing a reduction of 179 bp in the CDS sequence. Inferred by DNAMAN and CD-search, 59 amino acid residues at the C-terminus of OsHO1 in yl(t) would be missed to impair the ability of heme binding. In addition, qRT-PCR analysigbbbs found several key genes involving in chlorophyll biosynthesis expressed differently in yl(t). The OsHemA (hemin A gene) that encodes glutamyl-tRNA reductase was up-regulated, while OsCAO1 (chlorophyllide a oxygenase 1 gene) and OsNOL (non-yellow coloring1-like gene) that encodes chlorophyll b reductase expressed lower. This study reveals yl(t) is an allele of OsHO1 to mutate at splicing site, and provides a new germplasm for further understanding of the biological function of OsHO1.

WRKY transcription factors play key roles in the responses against biotic or abiotic stress in plants. To elucidate the biological function of sweet potato (Ipomoea batatas) WRKY transcription factor IbWRKY61 and to clarify its role in the protein interaction network, in this study, the bait construct pGBKT7-DEST-IbWRKY61 was constructed using Gateway method and used to screen the sweet potato cDNA library via the yeast (Saccharomyces cerevisiae) two-hybrid (Y2H) library screening system. The results showed that 241 positive clones were obtained through Y2H screening. These clones were cultured and detected using PCR, and a total of 121 positive monoclones were selected and the PCR products were recovered from agarose gel. High thought-out sequencing was performed with mixed PCR products, and the obtained reads were mapped to genes using STAR software package. According to transcripts per million (TPM) values, the top 100 gene sequences were selected and blasted against NCBI database for annotation, and the results showed that screened interacting proteins of IbWRKY61 were mainly involved in biotic stress response and pathogen defense. The interaction of IbWRKY61 and 12 of the screened interacting proteins were validated using Y2H and bimolecular fluorescence complementation (BiFC). Y2H test confirmed the interaction between IbWRKY61 and all the 12 proteins, and BiFC results confirmed that IbWRKY61 interacted with CCR4-associated factor 1 (CAF1) in the nucleus of Nicotiana benthamiana mesophyll cells, and interacted with ubiquitin-activating enzyme E1, Clp protease proteolytic subunit-related protein 2 (CLP2) and leucine-rich repeat receptor-like protein kinase BAK1-interacting receptor-like kinase 1 (BIR1), in the plasma membrane, chloroplast, plasma membrane or plasmodesma, respectively. The present study provides a reference for further research on the biological function of WRKY transcription factors in stress response and disease resistance of sweet potato.

Apple tree canker caused by Valsa mali seriously restricts the development of apple industry in China. It is of great theoretical significance for the effective prevention and control of apple tree canker to comprehensively analyze the pathogenic molecular mechanism of V. mali. The pH-responsive transcription factor PacC play an important role in the infection and pathogenic process of V. mali. In order to reveal PacC transcriptional regulation mechanism, the gene expression profiles of V. mali wild-type 03-8 and the PacC deletion mutant ΔVmPacC on branches were analyzed by RNA-seq assays. The results showed that there were 238 and 449 differentially expressed genes (DEGs) during pre-infection and post-infection of V. mali. GO enrichment analysis showed that biological processes of DEGs were mainly involved in carbohydrate metabolic processes, oxidation-reduction process, transmembrane transport process and polysaccharide metabolic processes. KEGG functional enrichment analysis showed that the pathways of DEGs were mainly involved in starch and sucrose metabolism, cyanoamino acid metabolism, pentose and glucuronate interconversions, these DEGs involved in three pathways were mainly related to plant cell wall polysaccharide (PCWP) degradation. The amino acid sequences of DEGs involved PCWP degradation were submitted to Pathogen Host Interactions (PHI)-base blast, that obtained 3 potential pathogenic related genes including putative pectate lyase A gene, pectin lyase B gene, hypothetical protein gene VM1G_08430. In this study, six DEGs were selected for qRT-PCR verification, and the results showed that the mRNA expression patterns of the 6 genes were consistent with the results of RNA-seq assays. The comprehensive analysis showed that transcription factor PacC functions in the infection and pathogenic process by regulating the expression of PCWP degradation genes. The study revealed PacC transcriptional regulation mechanism in the infection and pathogenic process of V. mali, and it would lay a theoretical foundation a theoretical basis for explaining the pathogenic mechanism of V. mali.

Sulfur dioxygenase 1 (ethylmalonic encephalopathy protein 1, ETHE1), the key enzyme in the oxidation of sulfides into sulfates, is mainly involved in plant stress tolerance, hormone response, seed development and survival. In this study, two VvETHE1s were cloned from Vitis vinifera. 'Pinot Noir' through reverse transcription PCR (RT-PCR), and named VvETHE1A (GenBank No. LOC100248855) and VvETHE1B (GenBank No. LOC100262655). The ORF of VvETHE1A was 867 bp, encoding 288 amino acids; while VvETHE1B was 861 bp encoding 286 amino acids. Multiple sequence alignment indicated that VvETHE1s were highly conserved in the domain of metallo-β-lactamase family. Evolutionary analysis showed that the genetic distance between VvETHE1A and jujube (Ziziphus jujuba) ETHE1 was closer than VvETHE1B. By semi-quantitative reverse transcription and PCR (sqRT-PCR), tissue-specific expression analysis showed both VvETHE1A and VvETHE1B were expressed in all tissues with different levels, among which VvETHE1B was highly expressed in root. qRT-PCR analysis showed that the expression levels of VvETHE1A and VvETHE1B were significantly different in the ovules of Vitis vinifera 'Pinot noir' and 'Thompson Seedless'. And both of them decreased in the ovules of 35~45 d after full-bloom of Thompson Seedless. VvETHE1A responded strongly to exogenous abscisic acid (ABA), followed by ethylene (ETH), while VvETHE1B strongly responded to methyljasmonate (MeJA), weakly to ABA and ETH. Moreover, the prokaryotic expression vectors of VvETHE1s were constructed and 2 fusions of about 35 kD were induced in the form of inclusion body. Decrease of induction temperature and increase of time resulted in high yield of recombinant VvETHE1s. The optimal induction condition was 28 ℃ for 6 h, and more soluble proteins were induced at 16 ℃. This study provide a basis for further studies on the regulatory mechanism of VvETHE1s in seed abortion and hormone response, and on the physiological and biochemical characteristics of VvETHE1s.

Calcium-dependent protein kinases (CDPKs or CPKs) function as Ca²⁺ sensors and effectors to directly translate Ca²⁺ signals into downstream phosphorylation signals. CDPKs are involved in plant growth and developmental processes, as well as responses to abiotic and biotic stresses. However, little information is available about the CDPK family in Saccharum plants. In this study, based on the published Saccharum spontaneum genome data, the CDPK gene family members of S. spontaneum were identified by bioinformatics methods, and a total of 90 CDPKs were identified, whose physical and chemical parameters, chromosomal locations, gene duplication, phylogenetic relationships, evolutionary characteristics, gene structure, and motif distribution were analyzed. Syntenic analysis showed that consensuses in CDPKs might have existed before the species divergence between S. spontaneum and Oryza sativa . The selection pressure analysis showed that CDPK genes in S. spontaneum were positively selected in the process of evolution and might play important roles in the evolution of plants. Transcriptome analysis showed that most SsCDPKs exhibited different expression levels in different tissues and developmental stages. In addition, many SsCDPKs in sugarcane hybrid variety Yuetang 55 (YT55) exhibited different expression profile under low-potassium and low-nitrogen treatments. The diverse expression of SsCDPKs suggested potential important function of SsCDPKs in regulating the development and responses to low-potassium and low-nitrogen stresses of Saccharum plants. This study provides a reference for further analysis of roles of Saccharum CDPK genes in the development and responses to nutrition stresses.

Growth hormone receptor (GHR) plays an important role in animal growth and development. In order to study the expression of long noncoding RNAs (lncRNAs) related to GHR gene in Cong Jiang Xiang pig (Sus scrofa), this paper selected Guizhou Cong Jiang Xiang pig and Yorkshire pig as the research objects, used the growth and development related miR-146b-3p as correlation point, screened out the GHR gene related lncRNAs by SWchen 2.3 program, and verified the lncRNAs by cloning and sequencing. The candidate lncRNA NONSUST017828.1 (GenBank No. TCONS_00020564, named lncGHR1) was used as the further research object. Bioinformatics and tissue expression spectrum analysis were performed, the lncGHR1-CMV over-expression vector was constructed and transfected to human embryonic kidney cells (HEK-293T) and primary myocytes of Cong Jiang Xiang pig, effects of lncGHR1 on the expression of Yes-associated protein (YAP) and transcription activator (TAZ) were detected by qRT-PCR. The results showed that the minimum free energy of the secondary structure of lncGHR1 was -131.20 kcal/mol and contained 3 ORF. The results of local comparison showed that lncGHR1 was the most similar with GHR 3'UTR of pig. The inhibition coefficient of lncGHR1 binding to miRNA was 4.96%, and the score of miR-146-3p with lncGHR1 was the highest. The expression of lncGHR1 was the highest in the spleen and lung of Cong Jiang Xiang pig, which was significantly higher than that in other tissues (P<0.01), the lowest in the heart; the highest expression of Yorkshire was found in liver, followed by lung; the expression of lncGHR1 in heart, spleen, lung, kidney, large intestine, small intestine and longissimus dorsi muscle of Cong Jiang Xiang pig was significantly higher than that of Yorkshire pig (P<0.01), but the expression of lncGHR1 in liver was significantly lower (P<0.01). Over-expression of lncGHR1 in 293T cells and primary myocytes of Cong Jiang Xiang pig significantly increased the expression of TAZ gene (P<0.05), but the expression of YAP gene was not significantly different (P>0.05). These results indicated that lncGHR1 might play a key role in the process of cell proliferation and growth. The above results indicated that lncGHR1 played an important role in the growth and development, and the was significant for the analysis of the genetic mechanism of Cong Jiang Xiang pigs' slow growth and short stature as well as the genetic improvement breeding of pigs.

Myostatin (Mstn) gene is a well-studied and well-prospected muscle development inhibitory factor.Mstn gene has a wide range of endocrine characteristics, which not only regulate muscle development, but also regulate the metabolic processes such as glucose, lipids and amino acids. In this study, the Mstn gene-edited cattle (Bos taurus) and normal control were used to detect the changes of key metabolites in serum under the influence of exercise and feeding. The results showed that the fasting blood glucose of Mstn^-/+ cattle was significantly lower than that of the control group (P<0.05), After exercise, the blood glucose levels of Mstn^-/+ cattle and control cattle decreased significantly, and the Mstn gene mutation accelerated this changed. The serum cholesterol, triglyceride, high density lipoprotein, low density lipoprotein content changes relatively stable, neither exercise nor feeding could change the difference trend of Mstn^-/+ cattle and control cattle. The test results of free fatty acids in serum showed that under fasting state, exercise would significantly reduced the content of saturated fatty acids in Mstn^-/+ bovine serum and increased the level of unsaturated fatty acids (including monounsaturated fatty acids and polyunsaturated fatty acids). From the perspective of amino acid levels in serum, the Mstn mutation increased the concentration of essential and non-essential amino acids in bovine plasma, but exercise and food intake would not significantly affect the level of amino acids in serum. The results of this study have certain guiding significance for in-depth understanding of the function of Mstn gene and exploring the metabolic regulation mechanism of Mstn.

In order to explore the genetic effect of polymorphism in the bone morphogenetic protein receptor-IB (BMPR-IB) gene coding region on the reproductive performance of sheep (Ovis aries), ewes of 4 sheep breeds were used as the research object in this study, which includes 65 multiple Small Tail Han sheep (SM), 71 twins Mongolian sheep (MT), 70 singleton Mongolian sheep (MS), 52 twins Oula sheep (ZT), 51 singletons Oula sheep (ZS), and 74 singletons Dorper sheep (DS). Using PCR combined DNA direct sequencing method, the polymorphism of 597 loci in the coding region of BMPR-IB gene of different fertility ewes was tested, and the correlation analysis was performed with the litter size. The results showed that: G/A mutation was detected at the 597 site of the coding region of the BMPR-IB gene, GG, GA and AA genotypes were detected in each experimental group sheep. The dominant genotype in SM was AA, and the dominant allele was A. The dominant genotype in MT, MS, ZT and ZS was GA, the dominant allele was G. The dominant genotype in DS was GG, and the dominant allele was G. All the experimental group sheep were in moderate polymorphism (0.25<PIC<0.5); the litter size of homozygous mutation AA type was significantly higher than that wild GG type and heterozygous mutant GA type in the Oula sheep experimental population (P<0.05). Therefore, this locus can be used as a potential molecular genetic marker site for sheep lambing number. This study provides theoretical support for the genetic mechanism and breeding of sheep lambing traits.

Rearing in cages is the development trend of duck production. This study analyzed the effects of rearing patterns on slaughter performance and intestinal microbiota in duck (Anas platyrhynchos). 16S rRNA high-throughput sequencing technology was used to reveal the fecal microbiota structure and composition in male and female ducks rearing on the floor or in cages groups. Forty ducks were selected from different rearing patterns. The slaughter performance was measured. Their fresh feces were collected from each duck. Total microbial DNA was extracted from feces samples. High-throughput sequencing was performed and the sequences were analyzed by QIIME 1.9.1 software. The results showed: 1) The slaughter performance of Gaoyou ducks was high. 2) The microbiota abundance and diversity of male ducks were higher than female ducks in rearing in cages group, but the abundance and diversity of fecal microbiota in female ducks were higher than male ducks in rearing on the floor group. The absolute dominant phyla in Gaoyou duck feces were Firmicutes and Bacteroidetes, the absolute dominant genera were Bacteroides and Romboutsia. Although there were no significant difference, the relative abundance values of dominant phylum and genera were different among groups. There were 6 species whose average relative abundance was more than 1%, such as Clostridiales bacterium, there were 50% species belonged to Bacteroides. 3) Compared to other groups, Bacteroides caecigallinarum was the differential microorganism in the feces of male duck in rearing in cages group, its relative abundance was significantly correlated with the pectoral muscle rate. Generally, rearing patterns and sex both affected fecal microbiota structure and composition in ducks, and the rearing patterns showed different effects on microbiota between sex, the abundance and diversity of male ducks rearing in cages was high. Bacteroides caecigallinarum may play an important role in the intestinal microbiota in ducks. The above results revealed that intestinal microbiota of ducks were affected by multiple factors, and regulation of intestinal microbiota could improve the production performance of duck.

Fanjing mountain is the main peak of Wuling mountain range. It has rich amphibian diversity due to its diverse climate types and superior hydrothermal conditions. However, the related research mainly concentrated in the 1980's, and mostly based on field investigation. Therefore, it is urgent to evaluate the genetic diversity of amphibians in Fanjingshan region by using mitochondrial genes as analytical markers. To evaluate the genetic diversity of Fejervarya multistriata in the surrounding area of Fanjing mountain, the influence of the specific geographical environment of Fanjing mountain on its survival status, and to provide scientific basis for further protection and utilization of genetic resources of F. multistriata, the present study used partial sequences of mitochondrial DNA (mtDNA) 12S rRNA as molecular markers, 185 samples from 10 districts and counties in Fanjingshan area were sequenced and the genetic diversity was analyzed. The results showed that nucleotide diversity index Pi and haplotype diversity (Hd) were 0.029 42±0.000 34 and 0.563±0.017, respectively. Ten haplotypes were found, and the dominant haplotypes were haplotype 1 and 2, followed by haplotype 8, accounting for 36.76%, 44.32% and 12.43% of the total sample, respectively. Haplotype 1 and haplotype 8 were mainly composed of the populations east of Fanjing mountain, while haplotype 2 mainly existed in the population west of Fanjing mountain. Haplotype network mediation diagram showed that dominant haplotype 1 and haplotype 8 gathered into one class, and haplotype 2 gathered into another class. Hoplobatrachus chinensis and Nanorana ventripunctata were used as extranet to construct a haplotype systemic-occurrence tree, and the haplotype clustering analysis indicated that the haplotype cluster of Hoplobatrachus chinensis and F. multistriata were together, and the haplotype cluster of F. multistriata was divided into 2 subbranches, which was consistent with the results of haplotype network map and population phylogenetic tree, that was, the 10 populations of the F. multistriata could be divided into 2 branches as east and west of Fanjing mountain, indicating that it might be caused by the geographical isolation of Fanjing mountain vein. The fixed coefficient Fst indicated that the population differentiation degree was obvious in F. multistriata population. Analysis of molecular variance (AMOVA) showed that the genetic variation was mainly between populations (86.14%), while the genetic variation within populations was low (13.86%). Comprehensive analysis showed that the genetic diversity among different geographic populations of F. multistriata was relatively low, and genetic differentiation was obvious, which might be mainly due to the geographical isolation of complex landforms and mountains. The above results have some reference value for further research on the influence of complex geographical environment and climatic characteristics of Fanjing moutain area (such as the obvious vertical climate zone spectrum) on other amphibians.

Porcine rotavirus (PoRV) is a member of the Reovirus family, belonging to the genus Rotavirus. PoRV is a widely prevalent zoonotic disease in the world. At present, developing a safe and effective vaccine is one of the important means to control rotavirus infection. The aim of this study was to express Porcine rotavirus VP7 yeast protein and to identify its immunological properties. First, the primers were designed according to the full sequence of VP7 published in GenBank (NC_011503.2), and established recombinant yeast engineering strain with fusion expression vector psep-2-VP7 from the G-2 proton containing VP7 to amplification of the purpose of the fragment, isopropyl-β-D-sulfur semi-lactose glycoside (isopropyl β-D-thiogalactoside, IPTG) induces the expression of glutathione-S-transferase (GST)-VP7 fusion protein, after which the optimized fermentation process conditions of the fusion protein GST-VP7 were isolated and purified and immunoimmune to mice (Mus musculus) for immunogenic analysis. The results showed that the size of the cloned VP7 gene was consistent with the expected glycoprotein gene, and the molecular weight of the recombinant protein was about 61 kD expressed in millet merozoa. After the fermentation conditions were optimized, the protein concentration was up to 0.99 mg/mL. The experimental results showed that after 2 weeks of VP7 protein immunization, the average geometric titer of IgG antibody in the nasal drip (IN) and intramuscular injection groups (IM) reached 495 and 680, respectively, and the average geometric titer of SIgA IN the nose drop group reached 43.5, which could produce high level of γ-interferon (IFN-γ), indicating that VP7 protein immunity could effectively induce the body to produce cellular immunity. The eukaryotic expression of VP7 protein in this study provided conditions for the follow-up study of this protein. The study on the immunogenicity of this protein has laid a foundation for the study on diagnostic antigens of rotavirus and related vaccines.

Gene editing is a technology that precisely modifies endogenous genes in living organisms. From its appearance to the application, it has greatly promoted the development of agricultural science. The development of gene editing technology is divided into three main stages: the zinc finger nuclease (ZFN) stage, the transcription activation-like effector nuclease (TALEN) stage, and the clustered regularly interspaced short palindromic repeats (CRISPR) stage. Gene editing technology has the characteristics of specifically recognizing target sequences and targeted modification of targeted genes. This technology is applied to the verification of the main genes of key traits of agricultural animals, and the breeding process of agricultural animals is accelerated through precise editing of known functional genes. This review will focus on gene editing and its application in agricultural animals, and provide references for research in animal breeding and related fields.

As an excellent renewable fuel, butanol is of great value to solve the problem of shortage of petroleum resources. Clostridium beijerinckii is the most important strain for butanol production. In this paper, the latest research progress of butanol production by C. beijerinckii was reviewed from four aspects: strain selection and molecular transformation to improve the butanol production and enhance the resistance to the environment, the development and utilization of cheap substrates, the optimization of butanol fermentation conditions and the improvement of production process, and the co-culture with cellulose or oxygen treatment bacteria to promote butanol production. This review provides a reference for the green and efficient production of butanol by C. beijerinckii.

The herbicide resistance and Lepidopteran resistance of rice (Oryza sativa) play important roles in weeds management and reduction of yield loss by insect pests, and the event-specific detection method is an essential technique for supervision of transgenic rice. In this study, the expression vector was constructed firstly, in which the Lepidopteran resistance gene Cry1Ca^# (gene coding insecticidal crystal protein Cry1Ca) was driven by wound-induced promoter and the herbicide resistance gene Epsps^# (gene coding 5-enolpyruvylshikimate 3-phosphate synthase) was driven by constitutive promoter, and a single copy transgenic event E1C9K-18 was developed by Agrobacterium-mediated genetic transformation from 9K19-5 (O. sativa subsp. indica), which possessed elite glyphosate resistance. Then, the hiTAIL-PCR (high-efficiency thermal asymmetric interlaced PCR) was adopted to reveal the flanking sequence of transgenic rice E1C9K-18, and the right flanking sequence with 420 bp length was discovered. Comparing the right flanking sequence with rice genome database, it was found the exogenous fragment was inserted behind the nucleotide residue No. 33 189 510 of chromosome 4. The left flanking sequence with 613 bp length was amplified using the primers that were designed according to the sequence of integration site on rice genome and left side sequence of T-DNA. Compared the left flanking sequence with rice genome database, it was found the exogenous fragment was inserted before the nucleotide residue No. 33 189 480 of chromosome 4. However, the insertion of T-DNA resulted in a deletion of 29 nucleotide residues in rice genome. Based on the left and right flanking sequences, the event-specific PCR detection method and tri-primers PCR method were developed for E1C9K-18, which will provide technical supports for identification of this transgenic event and rapidly selection of homozygote from segregation population.

False-negative result is a major factor affecting the results of nucleic acid testing. The internal amplification control (IAC) plays an important role in monitoring false negative results during PCR. In order to solve the false-negative result, the application of the IAC in reverse transcription- recombinase polymerase amplification (RT-RPA) was established. The IAC was constructed by the composite primer method and applied to the detection of three soybean viruses, Soybean mosaic virus (SMV), Bean pod mottle virus (BPMV) and Southern bean mosaic virus (SBMV), during RT-RPA. The results showed that the methods were able to achieve specific amplification of the target gene while indicating false negative results. Sensitivity experiments showed that the detection limit of this assay for SMV and BPMV was 0.05 ng, when the content of the IAC was 1.83×10⁴ copies in 50 μL reaction system. And when the content of the IAC was 1.83×10³ copies in 50 μL reaction system, the detection limit of this assay for SBMV was 0.05 ng. The methods were used to test 10 soybean (Glycine max) samples and the results were consistent with those of the corresponding reverse transcription-PCR (RT-PCR), indicating its good applicability. In conclusion, the IAC-RT-PRA methods for SMV, BPMV and SBMV were established in this study could indicate false negatives and improve credibility of test results.

Crop yield is determined by its genetics and stress resistance. Currently stress resistance breeding mainly uses plants as test objects, which is subject to environmental constraints and long test cycles. In order to find a convenient and scientific method for assessing plant stress resistance, this study took the strategy of using living cells to characterize plant. Tobacco (Nicotiana tabacum) BY-2 cells were adhered to the gold electrode surface of 5 MHz Quartz Crystal Microbalance with Dissipation (Q-sense) by using poly dimethyl diallyl ammonium chloride (PDADMAC) as electrostatic adsorbent, and the viscoelastic responses of BY-2 cells under stresses of 5%~20% PEG6000 were monitored in real time by Q-sense. The results showed that PDADMAC could be used as an adhesion agent for tobacco BY-2 cells, the Q-sense's dissipation increased and frequency decreased during the adhesion of BY-2 cells; the cells became softer under the stresses of PEG6000, and the cytoskeleton was softer than the cell wall. The Q-sense's responses were the most obvious at the moments when the cells were exposed to PEG6000, which was also affected by the PEG6000 concentration and the treatment time. Under the stress of 5% PEG6000, the cells continued to harden after a transient softening; while under the stresses of 15% and 20% PEG6000, the cells only experienced a brief hardening after the transient softening followed by a slightly further hardening (15% PEG6000) or no longer hardening (20% PEG6000). In addition, the Q-sense technique was used to compare the responses of BY-2 cells subjected to the treatments of 0%~20% PEG6000 concentration gradient and the subsequent 20%~0% PEG6000 inverse concentration gradient, the general trend was that the cells became gradually softer under the stresses of normal concentration gradient then the cells became gradually harder under the treatment of reverse concentration gradient. This study assessed the effect of PEG6000 stress on the viscoelastic changes of tobacco BY-2 cells in a real-time and quantitative manner, providing a new way for studying plant drought stress which would be helpful for the identification and evaluation of resistance germplasm at the cellular level.