Soil salinity affects the expression of serine/arginine-rich (SR) genes and isoforms by alternative splicing, which in turn regulates the adaptation of plants to stress.  We previously identified the cassava spliceosomal component 35 like (SCL) and SR subfamilies, belonging to the SR protein family, which are extensively involved in responses to abiotic stresses.  However, the post-transcriptional regulatory mechanism of cassava arginine/serine-rich (RS) subfamily in response to salt stress remains to be explored.  In the current study, we identified 37 genes of the RS subfamily from 11 plant species and systematically investigated the transcript levels of the RS40 and RS31 genes under diverse abiotic stress conditions.  Subsequently, an analysis of the conserved protein domains revealed that plant RS subfamily genes were likely to preserve their conserved molecular functions and played critical functional roles in responses to abiotic stresses.  Importantly, we found that overexpression of MeRS40 in Arabidopsis enhanced salt tolerance by maintaining reactive oxygen species homeostasis and up-regulating the salt-responsive genes.  However, overexpression of MeRS40 gene in cassava reduced salt tolerance due to the depression of its endogenous gene expression by negative autoregulation of its own pre-mRNA.  Moreover, the MeRS40 protein interacted with MeU1-70Ks (MeU1-70Ka and MeU1-70Kb) in vivo and in vitro, respectively.  Therefore, our findings highlight the critical role of cassava SR proteins in responses to salt stress in plants. 

In mammals, microRNAs (miRNAs) play key roles in multiple biological processes by regulating the expression of target genes.  Studies have found that the levels of miR-370-5p expression differ significantly in the skins of sheep with different hair colors; however, its function remains unclear.  In this study, we investigated the roles of miR-370-5p in sheep melanocytes and found that the overexpression of miR-370-5p significantly inhibited cell proliferation (P<0.01), tyrosinase activity (P=0.001) and significantly reduced (P<0.001) melanin production.  Functional prediction revealed that the 3´-untranslated region (UTR) of MAP3K8 has a putative miR-370-5p binding site, and the interaction between these two molecules was confirmed using luciferase reporter assays.  In situ hybridization assays revealed that MAP3K8 is expressed in the cytoplasm of melanocytes.  The results of quantitative RT-PCR and Western blotting analyses revealed that overexpression of miR-370-5p in melanocytes significantly inhibits (P<0.01) MAP3K8 expression via direct targeting of its 3´ UTR.  Inhibition of MAP3K8 expression by siRNA-MAP3K8 transfection induced a significant inhibition (P<0.01) of melanocyte proliferation and significant reduction (P<0.001) in melanin production, which is consistent with our observations for miR-370-5p.  Target gene rescue experiments indicated that the expression of MAP3K8 in melanocytes co-transfected with miR-370-5p and MAP3K8-cDNA (containing sites for the targeted binding to miR-370-5p) was significantly rescued (P≤0.001), which subsequently promoted significant increases in cell proliferation (P<0.001) and melanin production (P<0.01).  Collectively, these findings indicate that miR-370-5p plays a functional role in inhibiting sheep melanocyte proliferation and melanogenesis by downregulating the expression of MAP3K8.  

With the implementation of the C-strain vaccine, classical swine fever (CSF) has been under control in China, which is currently in a chronic atypical epidemic situation.  African swine fever (ASF) emerged in China in 2018 and spread quickly across the country. It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety.  Atypical porcine pestivirus (APPV) was first detected in Guangdong Province, China, in 2016, which mainly harms piglets and has a local epidemic situation in southern China.  These three diseases have similar clinical symptoms in pig herds, which cause considerable losses to the pig industry.  They are difficult to be distinguished only by clinical diagnosis.  Therefore, developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential.  In this study, three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV (5´ UTR), African swine fever virus (ASFV) (B646L), and APPV (5´ UTR), followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay.  The results showed that the method did not cross-react with other swine pathogens (porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), pseudorabies virus (PRV), porcine parvovirus (PPV), and bovine viral diarrhea virus BVDV).  The sensitivity results showed that CSFV, ASFV, and APPV could be detected as low as 1 copy mL^–1; the repeatability results showed that the intra-assay and inter-assay coefficient of variation of ASFV, CSFV, and APPV was less than 1%.  Twenty-two virus samples were detected by the multiplex real-time PCR, compared with national standard diagnostic and patented method assay for CSF (GB/T 27540–2011), ASF (GB/T 18648–2020), and APPV (CN108611442A), respectively.  The sensitivity of this triple real-time PCR for CSFV, ASFV, and APPV was almost the same, and the  compliance results were the same (100%).  A total of 451 clinical samples were detected, and the results showed that the positive rates of CSFV, ASFV, and APPV were 0.22% (1/451), 1.3% (6/451), and 0% (0/451), respectively.  This assay provides a valuale tool for rapid detection and accurate diagnosis of CSFV, ASFV, and APPV.

In this study, we tested the ability of sodium dehydroacetate (SD) to extend the shelf-life of ‘Kyoho’ grape.  Among the different concentrations of SD tested (0, 0.01, 0.1 and 1.0 mmol L^–1), 0.01 mmol L^–1 SD was the most effective in prolonging the shelf-life of ‘Kyoho’ grape.  Compared with the control, the weight loss rate, browning index and hydrogen peroxide (H₂O₂) and malonaldehyde contents were significantly lower in the 0.01 mmol L^–1 SD treatment, whereas the healthy berry rate, berry firmness, total soluble solids (TSS) content, ascorbic acid content and superoxide dismutase (SOD) activity were significantly higher.  In addition, an analysis of ‘Kyoho’ grape DNA using methylation sensitive amplification polymorphism (MSAP) markers showed that the average DNA methylation level was significantly higher in the 0.01 mmol L^–1 SD treatment than in the control.  Together, these results indicate that 0.01 mmol L^–1 SD could be used to extend the shelf-life of ‘Kyoho’ grape.  Moreover, a strong connection between reactive oxygen species (ROS) metabolism and DNA methylation change during storage was revealed.

High yields of wheat are mainly obtained through a high level of nitrogen and irrigation supplementation.  However, excessive nitrogen and irrigation supplication increase the risk of lodging.  The main objectives of this work were to clarify the capacity of lodging resistance of wheat in response to nitrogen and irrigation, as well as to explore the effective ways of improving lodging resistance in a high-yield wheat cultivar. In this study, field experiments were conducted in the 2015–2016 and 2016–2017 growing seasons.  A wheat cultivar Jimai 22 (JM22), which is widely planted in the northern of Huang-Huai winter wheat region, was grown at Tai’an, Shandong Province, under three nitrogen rates and four irrigation treatments.  The lodging risk was increased with increased nitrogen rate, as indicated by increasing lodging index (LI) and lodging rate across both growing seasons.  With nitrogen increasing, the plant height, the basal internode length and the center of gravity height, which were positively correlated with LI, increased significantly.  While the density of the basal 2nd internode (for culm and leaf sheath) and cell wall component contents, which were negatively correlated with LI, decreased conspicuous along with nitrogen increased.  Increasing irrigation supplementation increased the 2nd internode culm wall thickness, breaking strength and leaf sheath density within limits which increased stem strength.  Among the treatments, nitrogen application at a rate of 240 kg ha^–1 and irrigation application at 600 m3 ha^–1 at both the jointing and anthesis stages resulted in the highest yield and strongest stem.  A suitable plant height ensures sufficient biomass for high yield, and higher stem stiffness, which was primarily attributed to thicker culm wall, greater density of the culm and leaf sheaths and higher cell wall component contents are the characteristics that should be taken into account to improving wheat lodging resistance.

Spring regrowth is an important trait for perennial plants including alfalfa, the most cultivated forage legume worldwide.  However, the genetic and genomic basis of the trait is largely unknown in alfalfa due to its complex genetic background of the tetroploid genome.  The objective of this study was to identify quantitative trait loci (QTLs) associated with spring regrowth using high-resolution genetic linkage maps we constructed previously.  In total, 36 significant additive effect QTLs for the trait were detected.  Among them, 10 QTLs individually explained more than 10% of the phenotypic variation (PVE) with four in P1 and six in P2.  Six overlapped QTLs intervals were detected with two and four intervals distributed in P1 and P2, respectively.  In P1, both overlapped genomic regions were located on homolog 7D.  In P2, the four QTLs with PVE>10% were co-localized on homolog 6D.  Meanwhile, six pairs of significant epistatic QTLs were identified in P2.  Screening of potential candidate genes associated with four overlapped QTLs (qCP2019-8, qLF2019-5, qLF2020-4, and qBLUP-3) narrowed down one candidate annotated as MAIL1.  The Arabidopsis homolog gene has been reported to play an important role in plant growth.  Therefore, the detected QTLs are valuable resources for genetic improvement of alfalfa spring vigor using marker-assisted selection (MAS), and further identification of the associated genes would provide insights into genetic control of spring regrowth in alfalfa.

Sucrose synthases (SUS) are a family of enzymes that play pivotal roles in carbon partitioning, sink strength and plant development.  A total of 11 SUS genes have been identified in the genome of Malus domestica (MdSUSs), and phylogenetic analysis revealed that the MdSUS genes were divided into three groups, named as SUS I, SUS II and SUS III, respectively.  The SUS I and SUS III groups included four homologs each, whereas the SUS II group contained three homologs.  SUS genes in the same group showed similar structural characteristics, such as exon number, size and length distribution.  After assessing four different tissues, MdSUS1s and MdSUS2.1 showed the highest expression in fruit, whereas MdSUS2.2/2.3 and MdSUS3s exhibit the highest expression in shoot tips.  Most MdSUSs showed decreased expression during fruit development, similar to SUS enzyme activity, but both MdSUS2.1 and MdSUS1.4 displayed opposite expression profiles.  These results suggest that different MdSUS genes might play distinct roles in the sink-source sugar cycle and sugar utilization in apple sink tissues.

The aim of this research was to study the influence of chlorsulfuron residue and cadmium on the enzymatic activity and photosynthetic apparatus of maize (Zea mays L.) plants.  Chlorsulfuron and cadmium at 0.001 and 5.0 mg kg^–1, respectively, were mixed and applied to soil prior to planting.  The levels of chlorsulfuron- and cadmium-induced stress to plants were estimated by growth, chlorophyll content, lipid peroxide content, enzyme activities, and major fluorescence parameters of chlorophyll (revealed by the fluorescence imaging system FluorCam).  Chlorsulfuron negatively affected the chlorophyll content, photochemical efficiency of photosystem II in the dark-adapted state, the maximum efficiency of photosystem II, photochemical quenching coefficient, and steady-state fluorescence decline ratio in the leaves of maize seedlings.  However, cadmium did not produce noticeable changes.  Plants that were exposed to both chlorsulfuron and cadmium showed an obvious increase in the steady-state fluorescence decline ratio.  These results implied that the seedlings possessed more resistance to cadmium than to chlorsulfuron and their resistance to chlorsulfuron toxicity was enhanced by the presence of cadmium.  The results also suggested that chlorophyll fluorescence imaging reveals overall alterations within the leaves but may not reflect small-scale effects on tissues, as numeric values of specific parameters are averages of the data collected from the whole leaf.

Heat stress seriously affects wheat production in many regions of the world.  At present, heat tolerance research remains one of the least understood fields in wheat genetics and breeding and there is a lack of effective methods to quantify heat stress and heat tolerance in different wheat cultivars.  The objective of this study was to use various wheat cultivars to evaluate stress intensity (δ) and a new method for quantification of heat tolerance and compare this technique with three other currently utilized methods.  This new parameter for heat tolerance quantification is referred to as the heat tolerance index (HTI) and is an indicator of both yield potential and yield stability.  Heat treatments were applied in a controlled setting when anthesis had been reached for 80% of the wheat.  The stress intensity evaluation indicated heat shock was the main factor associated with kernel weight reduction while grain yield reduction was mainly associated with chronic high temperature.  The methods evaluation showed that a temperature difference of 5°C from natural temperatures was a suitable heat treatment to compare to the untreated controls.  HTI was positively correlated with yield under heat stress (r=0.8657, δ₂₀₁₀=0.15, in 2009–2010; r=0.8418, δ₂₀₁₁=0.20, in 2010–2011; P<0.01), and negatively correlated with yield reduction rate (r=–0.8344, in 2009–2010; r=–0.7158, in 2010–2011; P<0.01).  The results of this study validated the use of HTI and temperature difference control for quantifying wheat heat tolerance that included the yield potential and the stability of different wheat cultivars under heat stress.  Additionally, 10 wheat cultivars showed high HTI and should be further tested for their heat confirming characteristics for use in wheat heat tolerance breeding.

Ovine bones are the major by-products after slaughtered.  The present study was conducted to extract and characterize acid soluble collagens (ASC) and pepsin soluble collagens (PSC) from ovine bones (Ujumuqin sheep).  Ovine bones collagen were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) as type I collagen.  The results of Fourier transform infrared (FTIR) spectra analysis testified the existence of triple superhelical structure in both ASC and PSC, showing pepsin did not disrupt the triple helical structure of ovine bones collagen.  Glycine, accounting for one-third of total amino acids, was the major amino acid for ovine bones collagen.  Higher imino acid content was responsible for higher thermal denaturation temperature of ovine bones collagen compared to fish collagens.  The isoelectric point of ASC was lower than PSC due to the higher content of acidic amino acids.  Therefore, this study provides the potential reference for collagen extraction and application of ovine bones by-procduct.

Carex rigescens (Franch.) V. Krecz is a wild turfgrass perennial species in the Carex genus that is widely distributed in salinised areas of northern China.  To investigate genome-wide salt-response gene networks in C. rigescens, transcriptome analysis using high-throughput RNA sequencing on C. rigescens exposed to a 0.4% salt treatment (Cr_Salt) was compared to a non-salt control (Cr_Ctrl).  In total, 57 742 546 and 47 063 488 clean reads were obtained from the Cr_Ctrl and Cr_Salt treatments, respectively.  Additionally, 21 954 unigenes were found and annotated using multiple databases.  Among these unigenes, 34 were found to respond to salt stress at a statistically significant level with 6 genes up-regulated and 28 down-regulated.  Specifically, genes encoding an EF-hand domain, ZFP and AP2 were responsive to salt stress, highlighting their roles in future research regarding salt tolerance in C. rigescens and other plants.  According to our quantitative RT-PCR results, the expression pattern of all detected differentially expressed genes were consistent with the RNA-seq results.  Furthermore, we identified 11 643 simple sequence repeats (SSRs) from the unigenes.  A total of 144 amplified successfully in the C. rigescens cultivar Lüping 1, and 69 of them reflected polymorphisms between the two genotypes tested.  This is the first genome-wide transcriptome study of C. rigescens in both salt-responsive gene investigation and SSR marker exploration.  Our results provide further insights into genome annotation, novel gene discovery, molecular breeding and comparative genomics in C. rigescens and related grass species.

Alfalfa (Medicago sativa L.) is an important forage crop and is also a target of many fungal diseases including Fusarium spp.  As of today, very little information is available about molecular mechanisms that contribute to pathogenesis and defense responses in alfalfa against Fusarium spp. and specifically against Fusarium proliferatum, the causal agent of alfalfa root rot.  In this study, we used a proteomic approach to identify inducible proteins in alfalfa during a compatible interaction with F. proliferatum strain YQC-L1.  Samples used for the two-dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF mass spectrometry were from roots and leaves of alfalfa cultivar AmeriGraze 401+Z and WL656HQ.  Plants were grown in hydroponic conditions and at 4 days post inoculation with YQC-L1.  Our disease symptom assays indicated that AmeriGraze 401+Z  was tolerant to YQC-L1 infection while WL656HQ was highly susceptible.  Analysis of differentially expressed proteins found in the 2-DE was further characterized using the MASCOT MS/MS ion search software and associated databases to identify multiple proteins that might be involved in F. proliferatum resistance.  A total of 66 and 27 differentially expressed proteins were found in the roots and leaves of the plants inoculated with YQC-L1, respectively.  These identified proteins were placed in various categories including defense and stress response related metabolism, photosynthesis and protein synthesis.  Thirteen identified proteins were validated for their expressions by quantitative reverse transcription (qRT)-PCR.  Our results suggested that some of the identified proteins might play important roles in alfalfa resistance against Fusarium spp.  These finding could facilitate further dissections of molecular mechanisms controlling root rot disease in alfalfa and potentially other legume crops.   

   Straw return is an important management tool for tackling and promoting soil nutrient conservation and improving crop yield in Huang-Huai-Hai Plain, China. Although the incorporation of maize straw with deep plowing and rotary tillage practices are widespread in the region, only few studies have focused on rotation tillage. To determine the effects of maize straw return on the nitrogen (N) efficiency and grain yield of winter wheat (Triticum aestivum L.), we conducted experiments in this region for 3 years. Five treatments were tested: (i) rotary tillage without straw return (RT); (ii) deep plowing tillage without straw return (DT); (iii) rotary tillage with total straw return (RS); (iv) deep plowing tillage with total straw return (DS); (v) rotary tillage of 2 years and deep plowing tillage in the 3rd year with total straw return (TS). Treatments with straw return increased kernels no. ear^–1, thousand-kernel weight (TKW), grain yields, ratio of dry matter accumulation post-anthesis, and nitrogen (N) efficiency whereas reduced the ears no. ha^–1 in the 2011–2012 and 2012–2013 growing seasons. Compared with the rotary tillage, deep plowing tillage significantly increased the grain yield, yield components, total dry matter accumulation, and N efficiency in 2013–2014. RS had significantly higher straw N distribution, soil inorganic nitrogen content, and soil enzymes activities in the 0–10 cm soil layer compared with the DS and TS. However, significantly lower values were observed in the 10–20 and 20–30 cm soil layers. TS obtained approximately equal grain yield as DS, and it also reduced the resource costs. Therefore, we conclude that TS is the most economical method for increasing grain yield and N efficiency of winter wheat in Huang-Huai-Hai Plain.

    A survey of the population densities of rice planthoppers is important for forecasting decisions and efficient control. Traditional manual surveying of rice planthoppers is time-consuming, fatiguing, and subjective. A new three-layer detection method was proposed to detect and identify white-backed planthoppers (WBPHs, Sogatella furcifera (Horváth)) and their developmental stages using image processing. In the first two detection layers, we used an AdaBoost classifier that was trained on a histogram of oriented gradient (HOG) features and a support vector machine (SVM) classifier that was trained on Gabor and Local Binary Pattern (LBP) features to detect WBPHs and remove impurities. We achieved a detection rate of 85.6% and a false detection rate of 10.2%. In the third detection layer, a SVM classifier that was trained on the HOG features was used to identify the different developmental stages of the WBPHs, and we achieved an identification rate of 73.1%, a false identification rate of 23.3%, and a 5.6% false detection rate for the images without WBPHs. The proposed three-layer detection method is feasible and effective for the identification of different developmental stages of planthoppers on rice plants in paddy fields.

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is one of the most severe wheat diseases.  Mining powdery mildew resistance genes in wheat cultivars and their appliance in breeding program is a promising way to control this disease.  Genetic analysis revealed that a single dominant resistance gene named PmTm4 originated from Chinese wheat line Tangmai 4 confers resistance to prevailing isolates of B. graminis f. sp. tritici isolate E09.  Detailed comparative genomics analyses helped to develop closely linked markers to PmTm4 and a fine genetic map was constructed using large F₂ population, in which PmTm4 was located into a 0.66-cM genetic interval.  The orthologous subgenome region of PmTm4 in Aegilops tauschii was identified, and two resistance gene analogs (RGA) were characterized from the corresponding sequence scaffolds of Ae. tauschii draft assembly.  The closely linked markers and identified Ae. tauschii orthologs in the mapping interval provide an entry point for chromosome landing and map-based cloning of PmTm4.

Pre-harvest sprouting (PHS) occurs frequently in most of the wheat cultivation area worldwide, which severely reduces yield and end-use quality, resulting in substantial economic loss.  In this study, quantitative trait loci (QTL) for PHS resistance were mapped using an available high-density single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) genetic linkage map developed from a 269 recombinant inbred lines (RILs) population of Yanda 1817×Beinong 6.  Using phenotypic data on two locations (Beijing and Shijiazhuang, China) in two years (2012 and 2013 harvesting seasons), five QTLs, designated as QPhs.cau-3A.1, QPhs.cau-3A.2, QPhs.cau-5B, QPhs.cau-4A, and QPhs.cau-6A, for PHS (GP) were detected by inclusive composite interval mapping (ICIM) (LOD≥2.5).  Two major QTLs, QPhs.cau-3A.2 and QPhs.cau-5B, were mapped on 3AL and 5BS chromosome arms, explaining 6.29–21.65% and 4.36–5.94% of the phenotypic variance, respectively.  Precise mapping and comparative genomic analysis revealed that the TaVp-1A flanking region on 3AL is responsible for QPhs.cau-3A.2.  SNP markers flanking QPhs.cau-3A.2 genomic region were developed and could be used for introgression of PHS tolerance into high yielding wheat varieties through marker-assisted selection (MAS).

Grain size is a major determinant of grain weight and a trait having important impact on grain quality in rice. The objective of this study is to detect QTLs for grain size in rice and identify important QTLs that have not been well characterized before. The QTL mapping was first performed using three recombinant inbred line populations derived from indica rice crosses Teqing/IRBB lines, Zhenshan 97/Milyang 46, Xieqingzao/Milyang 46. Fourteen QTLs for grain length and 10 QTLs for grain width were detected, including seven shared by two populations and 17 found in one population. Three of the seven common QTLs were found to coincide in position with those that have been cloned and the four others remained to be clarified. One of them, qGS10 located in the interval RM6100–RM228 on the long arm of chromosome 10, was validated using F2:3 populations and near isogenic lines derived from residual heterozygotes for the interval RM6100–RM228. The QTL was found to have a considerable effect on grain size and grain weight, and a small effect on grain number. This region was also previously detected for quality traits in rice in a number of studies, providing a good candidate for functional analysis and breeding utilization.

    Temperature extremes represent an important limiting factor to plant growth and productivity. Low concentration of hydrogen sulfide (H₂S) has been proven to function in physiological responses to various stresses. The present study evaluated the effect of foliar application of wheat seedlings with a H₂S donor, sodium hydrosulfide (NaHS), on the response to acute heat stress. The results showed that pretreatment with NaHS could promote heat tolerance of wheat seedlings in a dose-dependent manner. Again, it was verified that H₂S, rather than other sulfur-containing components or sodion derived from NaHS solution, should contribute to the positive role in promoting wheat seedlings against heat stress. To further study antioxidant mechanisms of NaHS-induced heat tolerance, superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11) activities, and H₂S, hydrogen peroxide (H₂O₂), malonaldehyde (MDA), and soluble sugar contents in wheat seedlings were determined. The results showed that, under heat stress, the activities of SOD, CAT, and APX, H₂S, H₂O₂, MDA, and soluble sugar contents in NaHS-pretreated seedlings and its control all increased. Meanwhile, NaHS-pretreated seedlings showed higher antioxidant enzymes activities and gene expression levels as well as the H₂S and soluble sugar levels, and lower H₂O₂, MDA contents induced by heat stress. While little effect was detected in antioxidant enzymes activities and soluble substances contents in pretreated wheat seedlings compared with its control under normal culture conditions (data not shown). All of our results suggested that exogenous NaHS could alleviate oxidative damage and improve heat tolerance by regulating the antioxidant system in wheat seedlings under heat stress.

    To speed up the degradation of corn stover directly returned to soil at low temperature, the corn stover-degrading microbial consortium GF-20, acclimated to biological decomposition in the frigid region, was successfully constructed under a long-term limiting substrate. To evaluate its potential in accelerating the decomposition of un-pretreated corn stover, the decomposing property, fermentation dynamic and the microbial diversity were analyzed. GF-20 degraded corn stover by 32% after 15-day fermentation at 10°C. Peak activities of filter paperlyase (FPA), β-glucosidases (CB), endoglucanases (Cx), and cellobiohydrolases (C1) were 1.15, 1.67, 1.73, and 1.42 U mL^–1, appearing at the 6th, 3rd, 11th, and 9th d, respectively. The pH averaged at 6.73–8.42, and the optical density (OD) value peaked at 1.87 at the 120 h of the degradation process. Cellulase, hemicellulase and lignin in corn stover were persistently degraded by 44.85, 43.85 and 25.29% at the end of incubation. Result of denaturing gradient gel electrophoresis (DGGE) profiles demonstrated that GF-20 had a stable component structure under switching the temperature and pH. The composition of the GF-20 was also analyzed by constructing bacterial 16S rDNA clone library and fungal 18SrDNA-PCR-DGGE. Twenty-two bacterial clones and four fungal bands were detected and identified dominant bacteria represented by Cellvibrio mixtus subsp., Azospira oryzae, Arcobacter defluyii, and Clostridium populeti and the fungi were mainly identified as related to Trichosporon sp.

    The vacuolar proton pump ATPase (V-H⁺-ATPase), which is a multi-subunit membrane protein complex, plays a major role in the activation of ion and nutrient transport and has been suggested to be involved in several physiological processes, such as cell expansion and salt tolerance. In this study, three genes encoding V-H⁺-ATPase subunits B (ScVHA-B, GenBank: JF826506), C (ScVHA-C, GenBank: JF826507) and H (ScVHA-H, GenBank: JF826508) were isolated from the halophyte Suaeda corniculata. The transcript levels of ScVHA-B, ScVHA-C and ScVHA-H were increased by salt, drought and saline-alkali treatments. V-H⁺-ATPase activity was also examined under salt, drought and saline-alkali stresses. The results showed that V-H⁺-ATPase activity was correlated with salt, drought and saline-alkali stress. Furthermore, V-H⁺-ATPase subunits B, C and H (ScVHA-B, ScVHA-C and ScVHA-H) from S. corniculata were introduced separately into the alfalfa genome. The transgenic alfalfa was verified by Southern and Northern blot analysis. During salt and saline-alkali stresses, transgenic linevacuolar proton pump, salt tolerance, saline-alkali tolerance, alfalfa
s carrying the B, C and H subunits had higher germination rates than the wild type (WT). More free proline, higher superoxide dismutase (SOD) activity and lower malondialdehyde (MDA) levels were detected in the transgenic plants under salt and saline-alkali treatments. Moreover, the ScVHA-B transgenic lines showed greater tolerance to salt and saline-alkali stresses than the WT. These results suggest that overexpression of ScVHA-B, ScVHA-C and ScVHA-H improves tolerance to salt and saline-alkali stresses in transgenic alfalfa.