病毒诱导的基因沉默（VIGS）和成簇规律间隔的短回文重复序列/CRISPR相关蛋白（CRISPR/Cas）系统是现代植物生物技术中快速、精准进行基因功能验证的有效手段。然而，关于萝卜基因沉默和编辑的研究仍然有限。在本研究中，利用烟草脆裂病毒（TRV）和芜菁黄化花叶病毒（TYMV）介导的基因沉默技术敲减萝卜中八氢番茄红素脱氢酶基因（RsPDS），获得具有白化表型的株系；且TYMV介导的基因沉默效率高于TRV。萝卜基因型‘NAU-067’叶片中RsPDS的表达水平显著下降。构建带有2个RsPDS靶序列的2300GN-Ubi-RsPDS-Cas9载体，采用发根农杆菌介导的遗传转化对‘NAU-067’无根苗进行侵染。结果发现毛状根中9条不定根经GUS染色呈现蓝色，Sanger测序进一步发现其中4条不定根在RsPDS基因的靶序列1上发生了编辑。此外，通过根癌农杆菌介导的萝卜子叶转化获得了白化株系。株系1在靶序列2处发生了5个碱基替换和3个碱基缺失，株系2在靶序列1处发生了3个碱基插入和3个碱基替换。利用VIGS和CRISPR/Cas9技术可以精准验证萝卜基因的生物学功能，因此，它将促进萝卜育种计划中重要园艺性状的遗传改良。

象耳豆根结线虫是一类在全球范围内发生且危害多种作物的重要病原线虫，其主要分布于全球的热带和亚热带地区。然而，目前关于影响其分布的主要环境因子以及未来潜在分布区域变化的研究还未有报道。在本研究中，我们根据象耳豆根结线虫在全球不同地区发生报道的相关数据，利用Maximum Entropy（MaxEnt）模型对这类根结线虫在中国及全球的潜在地域分布进行了预测。同时，我们利用3套气候模式（BCC-CSM2-MR, CanESM5和CNRM-CM6-1）对象耳豆根结线虫到本世纪50年代和90年代的潜在分布区域进行了预测，对其在不同气候条件下潜在分布区域的变化进行了分析。结果显示，象耳豆根结线虫的最佳适生区域为亚洲、南美洲、北美洲和非洲北纬30度到南纬30度之间的区域。Bio16（全年最湿润季度的降水量）、bio10（全年最热季度的平均气温）以及bio11（全年最冷季度的平均气温）是影响象耳豆根结线虫潜在分布最重要的环境因子。进一步的预测结果显示，在未来的气候条件下，全球象耳豆根结线虫的最佳适生区域将不断向高纬度地区扩展。该结果将为象耳豆根结线虫的防控提供理论基础。

本研究在前期已发掘短片段InDels和SNPs基础上，基于全基因组重测序分析在一个重组自交系群体的两个亲本中品03-5373（ZP）和中黄13（ZH）之间检测到不均匀分布在大豆20条染色体上的13573个长片段InDels，其中，Chr11上最少，有321个，Chr18上最多，有1246个。长片段InDels在染色体两臂的平均密度显著高于着丝粒区，与大豆基因组注释基因的分布模式一致。位于基因区的长片段InDels有2704个，占总数目的19.9%，其中319个为可导致蛋白质序列截短或延长的大效应InDels。重点对前期鉴定的株高相关QTL（qPH16）进行分析，共鉴定到35个长片段InDels，并将其开发成InDel标记，其中26个InDel标记（74.3%）在ZP和ZH之间表现出明显的多态性。利用开发的标记结合已有的4个SNPs标记对由ZP和ZH衍生的242个重组自交系进行基因型鉴定和QTL定位，将qPH16的定位区间从原来的960 kb缩小到477.55 kb，包含65个注释基因。在SNPs和短片段InDels开发基础上，进一步开发长片段InDels，可为大豆重要农艺性状的遗传分析和分子辅助选择育种提供更加全面的遗传变异信息。

大豆株高是由主效或微效基因控制的重要农艺性状。在已报道的株高QTL中，绝大部分定位区间较大，限制了大豆株高分子调控机制的解析。增加遗传图谱的标记密度会显著地提高QTL定位的效率和准确性。本研究利用双亲中黄13和中品03-5373及其衍生的241个重组自交系（RILs）全基因组重测序数据，构建一个包含4011个重组bin标记、总遗传距离为3139.15 cM的高密度遗传图谱，相邻bin标记间的平均距离为0.78 cM。比较基因组分析表明，所构建的遗传图谱与大豆参考基因组具有较高的共线性。基于此图谱，在6个环境中共检测到9个株高QTL，包括3个新位点（qPH-b_11，qPH-b_17和qPH-b_18）。其中，两个环境稳定主效QTL qPH-b_13和qPH-b_19-1可解释10.56%～32.7%的表型变异。qPH-b_13和qPH-b_19-1被精细定位到440.12 kb和237.06 kb的基因组区间，分别包含54和28个注释基因。进一步的拟南芥同源基因功能和候选基因表达分析表明，基因Glyma.13G292600和Glyma.19G194100分别为qPH-b_13和qPH-b_19-1的候选功能基因。

堆肥过程中磷矿粉影响微生物群落进而钝化重金属的机制相关研究较少。本研究通过分析猪粪秸秆堆肥过程中微生物群落结构及重金属形态变化，探明磷矿粉作用下微生物对重金属钝化机制。结果表明磷矿粉的添加能够提高堆肥过程中重金属的钝化效果，特别是对于Cd和Zn，生物有效性系数分别降低了247.41和176.25%。随着堆肥的进行，细菌群落结构发生变化，Firmicutes 所占比例降低，而Proteobacteria, Bacteroidetes, Deinococcus-Thermus 和Gemmatimonadetes所占比例升高。磷矿粉的添加使原生的细菌门类发生变化，Bacteroidetes变化可以解释Zn，Cu和Cr的形态变化，而 Deinococcus-Thermus和Proteobacteria影响Zn, Pb和Cd钝化。该研究结果能为堆肥过程中的重金属钝化提供支持。

Curvularia leaf spot, caused mainly by Curvularia lunata, is a widespread plant disease in China.  In the recent years, directional host selection by the pathogen, which likely results in the virulence differentiation in pathogens, is widely reported.  Among the hallmarks potentially associated to pathogen variation in virulence, superoxide dismutase gene Sod has been found to be closely related to the enhancement of virulence.  In the present study, the full-length of Sod was obtained via Blastn alignment against GenBank and the whole genome of C. lunata.  In order to understand the role of Sod in the virulence variation in C. lunata, targeted gene disruption was performed to construct Sod mutants.  The cell wall degrading enzyme (CWDE) activities and toxin production of ΔSod were not distinctly different from wild-type strain CX-3 and its complon.  However, at an early stage of infection, ΔSod virulence appeared to be lower than CX-3 and the complon, while at a later stage, its virulence gradually returned to the level of CX-3 and the complon.  Furthermore, the melanin production of ΔSod was significantly reduced compared to CX-3 and the complon, suggesting that Sod gene influences the virulence by regulating melanin production at an early stage of infection but is not essential for pathogenicity.  However, the disruption of Sod did not significantly affect the transcriptional expression of the melanin biosynthesis-associated genes, brn1 and scd.  Therefore, we infer that Sod in C. lunata are involved, to some extent, with the virulence in maize leaf, but still needs further studies to have a clear understanding of its mechanism.

Nutrient deficiency stresses often occur simultaneously in soil.  Thus, it’s necessary to investigate the mechanisms underlying plant responses to multiple stresses through identification of some key stress-responsive genes.  Quantitative real-time PCR (qRT-PCR) is essential for detecting the expression of the interested genes, of which the selection of suitable reference genes is a crucial step before qRT-PCR.  To date, reliable reference genes to normalize qRT-PCR data under different nutrient deficiencies have not been reported in plants.  In this study, expression of ten candidate reference genes was detected in leaves and roots of rapeseed (Brassica napus L.) after implementing different nutrient deficiencies for 14 days.  These candidate genes, included two traditionally used reference genes and eight genes selected from an RNA-Seq dataset.  Two software packages (GeNorm, NormFinder) were employed to evaluate candidate gene stability.  Results showed that VHA-E1 was the highest-ranked gene in leaves of nutrient-deficient rapeseed, while VHA-G1 and UBC21 were most stable in nutrient-deficient roots.  When rapeseed leaves and roots were combined, UBC21, HTB1, VHA-G1 and ACT7 were most stable among all samples.  To evaluate the stabilities of the highest-ranked genes, the relative expression of two target genes, BnTrx1;1 and BnPht1;3 were further determined.  The results showed that the relative expression of BnTrx1;1 depended on reference gene selection, suggesting that it’s necessary to evaluate the stability of reference gene prior to qRT-PCR.  This study provides suitable reference genes for gene expression analysis of rapeseed responses to different nutrient deficiencies, which is essential for elucidation of mechanisms underlying rapeseed responses to multiple nutrient deficiency stresses.

The cultivated soybean (Glycine max (L.) Merr.) was distinguished from its wild progenitor Glycine soja Sieb. & Zucc. in growth period structure, by a shorter vegetative phase (V), a prolonged reproductive phase (R) and hence a larger R/V ratio. However, the genetic basis of the domestication of soybean from wild materials is unclear. Here, a panel of 123 cultivated and 97 wild accessions were genotyped using a set of 24 presence/absence variants (PAVs) while at the same time the materials were phenotyped with respect to flowering and maturity times at two trial sites located at very different latitudes. The major result of this study showed that variation at PAVs is informative for assessing patterns of genetic diversity in Glycine spp. The genotyping was largely consistent with the taxonomic status, although a few accessions were intermediate between the two major clades identified. Allelic diversity was much higher in the wild germplasm than in the cultivated materials. A significant domestication signal was detected at 11 of the PAVs at 0.01 level. In particular, this study has provided information for revealing the genetic basis of photoperiodism which was a prominent feature for the domestication of soybean. A significant marker-trait association with R/V ratio was detected at 14 of the PAVs, but stripping out population structure reduced this to three. These results will provide markers information for further finding of R/V related genes that can help to understand the domestication process and introgress novel genes in wild soybean to broaden the genetic base of modern soybean cultivars.

Male sterility induced by a chemical hybridization agent (CHA) is an important tool for utilizing crop heterosis. Leaves, especially the flag leaves, as CHA initial recipients play a decisive role in inducing male sterility. To investigate effects of different treatment times of CHA-SQ-1 used, morphological, biochemical and physiological responses of wheat flag leaves were detected in this study. CHA induced programmed cell death (PCD) as shown in terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) and DNA laddering analysis. In the early phase, CHA-SQ-1 triggered organelle changes and PCD in wheat leaves accompanied by excess production of reactive oxygen species (O2 -. and H2O2) and down-regulation of the activities of superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (POD). Meanwhile, leaf cell DNAs showed ladder-like patterns on agarose gel, indicating that CHA-SQ-1 led to the activation of the responsible endonuclease. The oxidative stress assays showed that lipid peroxidation was strongly activated and photosynthesis was obviously inhibited in SQ-1-induced leaves. However, CHA contents in wheat leaves gradually reduced along with the time CHA-SQ-1 applied. Young flags returned to an oxidative/antioxidative balance and ultimately developed into mature green leaves. These results provide explanation of the relations between PCD and anther abortion and practical application of CHA for hybrid breeding.

The adaptability of soybean to be grown at a wide range of latitudes is attributed to natural variation in the major genes and quantitative trait loci (QTLs) that control flowering time and maturity. Thus, the identification of genes controlling flowering time and maturity and the understanding of their molecular basis are critical for improving soybean productivity. However, due to the great effect of the major maturity gene E1 on flowering time, it is difficult to detect other small-effect QTLs. In this study, aiming to reduce the effect of the QTL, associated with the E1 gene, on the detection of other QTLs, we divided a population of 96 recombinant inbred lines (RILs) into two sub-populations: one with the E1 allele and another with the e1nl allele. Compared with the results of using all 96 recombinant inbred lines, additional QTLs for flowering time were identified in the sub-populations, two (qFT-B1 and qFT-H) in RILs with the E1 allele and one (qFT-J-2) in the RILs with the e1nl allele, respectively. The three QTLs, qFT-B1, qFT-H and qFT-J-2 were true QTLs and played an important role in the regulation of growth period. Our data provides valuable information for the genetic mapping and gene cloning of traits controlling flowering time and maturity and will help a better understanding of the mechanism of photoperiod-regulated flowering and molecular breeding in soybean.

Nitrogen (N) is one of the macronutrients required for plant growth, and reasonable application of N fertilizers can increase crop yields and improve their quality. However, excessive application of N fertilizers will decrease N use efficiency and also lead to increases in N2O emissions from agricultural soils and many other environmental issues. Research on the effects of different N fertilizer management practices on wheat yields and N2O emissions will assist the selection of effective N management measures which enable achieving high wheat yields while reducing N2O emissions. To investigate the effects of different N management practices on wheat yields and soil N2O emissions, we conducted field trials with 5 treatments of no N fertilizer (CK), farmers common N rate (AN), optimal N rate (ON), 20% reduction in optimal rate+dicyandiamide (ON80%+DCD), 20% reduction in optimal rate+nano-carbon (ON80%+NC). The static closed chamber gas chromatography method was used to monitor N2O emissions during the wheat growing season. The results showed that there were obvious seasonal characteristics of N2O emissions under each treatment and N2O emissions were mainly concentrated in the sowing- greening stage, accounting for 54.6–68.2% of the overall emissions. Compared with AN, N2O emissions were decreased by 23.1, 45.4 and 33.7%, respectively, under ON, ON80%+DCD and ON80%+NC, and emission factors were declined by 22.2, 66.7 and 33.3%, respectively. Wheat yield was increased significantly under ON80%+DCD and ON80%+NC by 12.3 and 11.9%, respectively, relative to AN while there was no significant change in yield in the ON treatment. Compared with ON, overall N2O emissions were decreased by 29.1 and 13.9% while wheat yields improved by 18.3 and 17.9% under ON80%+DCD and ON80%+NC, respectively. We therefore recommend that ON80%+DCD and ON80%+NC be referred as effective N management practices increasing yields while mitigating emissions.

The novel cry1Ai gene that cloned from Bacillus thuringiensis strain SC6H8 encoded a protein exhibiting strong toxicity against Plutella xylostella and Chilo suppressalis in our previous study. Using the available information for the active fragments of other Cry toxins, eight truncated fragments were constructed to identify the minimal active fragment of Cry1Ai. All truncated fragments were expressed in Escherichia coli strain BL21 (DE3), and the insecticidal activity against 2nd- instar P. xylostella larvae was assessed using full-length Cry1Ai as a positive control. The results indicate that the minimal active fragment of the Cry1Ai toxin against P. xylostella is located between amino acid residues 36I and 605I, which is smaller than the regions previously reported for Cry1A. The first two amino acids (34T and 35P) on helix α-1 and whole helix α-2 of domain I and sheet β-32 of domain III are necessary for Cry1Ai toxin to keep its toxicity against P. xylostella.

In order to establish double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for detection of duck or goose flavivirus, polyclonal antibody against the flavivirus strain JS804 in geese and monoclonal antibody against the E protein of flavivirus strain JS804 in geese were used as the capture antibody and detection antibody, respectively. The optimal dilution of the capture antibody and detecting antibody capable of detecting the flavivirus strain JS804 in geese were 1:3 200 and 1:160 in the check-board titration, respectively. The reaction time of sample was 1 h, and the optimal working dilution of HRP-labeled goat-anti-mouse IgG was 1:10 000. The positive standard value was 0.247 (OD450 nm). The geese flavivirus could be detected at a minimal concentration of 1.875 μg mL-1. The ELISA had no cross-reaction with Newcastle disease virus (NDV), Avian influenza virus (AIV), Infectious bronchitis virus (IBV), Infectious bursal disease virus (IBDV), Duck hepatitis virus (DHV), and Gosling plague virus (GPV). Twenty clinical samples were detected by the DAS-ELISA and RT-PCR respectively, with the agreement rate of 75%. The results revealed that the DAS-ELISA possessed favorable specificity and higher sensitivity, indicating a suitable method for rapid detection of the duck or goose flavivirus.

Supercritical carbon dioxide (SC-CO2) extraction was employed to extract oil from Nigella glandulifera Freyn seed in this study. Response surface methodology (RSM) was applied to evaluate the effects of the process parameters (pressure, temperature, and CO2 flow rate) on oil yield of N. glandulifera seed. A Box-Behnken design was used to optimize the extraction parameters. The analysis of variance indicated that the linear coefficients of pressure and CO2 flow rate, the quadratic term coefficients of pressure and temperature and the interactions between pressure and temperature, as well as temperature and CO2 flow rate, had significant effects on the oil yield (P<0.05). The optimal conditions to obtain the maximum oil yield from N. glandulifera seed were pressure 30.84 MPa, temperature 40.57°C, and CO2 flow rate 22.00 L h-1. Under these optimal conditions, the yield of oil was predicted to be 38.19%. The validation experiment results agreed with the predicted values. The fatty acid composition of N. glandulifera seed oil extracted using SC-CO2 was compared with that of oil obtained by Soxhlet method. The results showed that the fatty acid compositions of oil extracted by the two methods were similar. Identification of oil compounds with gas chromatography-mass spectrometry (GC-MS) showed that the contents of unsaturated fatty acids linoleic acid (48.30%), oleic acid (22.28%) and saturated fatty acids palmitic acid (16.65%), stearic acid (4.17%) were the most abundant fatty acids in seed oil from N. glandulifera.

The investigation was carried out to study the response of winter rapeseed to potassium (K) feritlization and the critical soil available K level for current winter rapeseed production in the Yangtze River Valley (YRV) of China. A total of 132 field experiments were conducted in fields of farmers in the major winter rapeseed growing areas in YRV in 2000/2001 and 2004/2005 to 2006/2007 during growing season. Results of these field experiments showed that the average rapeseed yield increment resulting from 100 kg K ha^-1 application was 358 kg ha^-1, an increase over the control CK (no K) of 18.0% in 2005/2006 and 2006/2007. The average internal use efficiency (IE) of K was higher in the CK treatment (21.9 kg grain, kg-1 K uptake) than in the +K (100 kg K ha^-1) treatment (17.7 kg grain, kg^-1 K uptake). Winter rapeseed required 68.1 kg of K to produce 1 000 kg seed. The recovery efficiency of K fertilizer in rapeseed production averaged 39.3%. The K balance was negative, with an average net removal of 117.6 kg K ha^-1 in the CK treatment annually, and 56.8 kg K ha^-1 in the +K treatment. The results indicated that there was a significant negative relationship between yield increments by K application and soil available K content. Based on the relative yield of CK/+K at 90% level, the critical level of soil available K(NH₄OA_c-extractable K) was 135 mg kg^-1.

This study was carried out to investigate the transfection effect of exogenous gene into plant protoplast cell mediated by polyethylenimine (PEI) nanovector, based on PEI gene delivery system in the field of medical science. PEI/DNA complexes were prepared by using PEI polymer to bind the plant expression plasmid, pCMl205-GFPn. The ability of PEI combining and protecting DNA was investigated by agarose gel electrophoresis retardation assay. The surface characteristics of PEI/DNA complexes were observed with transmission electron microscope. The transfection efficiency of Arabidopsis thaliana protoplasts mediated by PEI/DNA complexes at different N/P ratios was analyzed based on observation of transient expression of green fluorescent protein with confocal laser scanning microscope. PEI could bind and condense DNA, and form stable 100-200 nm PEI/DNA complexes when the proportion of PEl and DNA is in the range of 5:1-1:4. Transfection efficiency of PEI/DNA complexes increased with N/P ratios in range of N/P<5 and reached the highest at N/P=5, and began to decrease beyond N/P>5 as higher toxicity to cells. The transfection efficiency of PEI/DNA complexes at N/P=5 was higher than PEG. This study confirmed that PEI nanovector could effectively mediate foreign gene entering into A. thaliana protoplast cell to obtain transient expression, which may be developed as a hopeful and novel transgenic method combined with plant protoplast regeneration.