To understand the subcellular location and enzymatic characteristics, BnALS3 (acetolactate synthase from Brassica napus L. was investigated. A full length cDNA of BnALS3 gene was cloned from rapeseed line N131 by RT-PCR. The open reading frame of BnALS3 was 1 959 bp in length, which encoded a 652-amino acids sequence with a putative chloroplast transit peptide including 49 amino acid residues at N-terminal end. The transit peptide DNA was cloned for plant-expressed vector p35S::BnALS3-GFP construction. Transient expression from transformed protoplasts of Arabidopsis thaliana showed that BnALS3 protein was mainly located in chloroplast. Bacterial expression vector pCzn1-BnALS3 (without chloroplast transit sequence) was constructed and expressed in Escherichia coli strain BL21 (DE3). SDS-PAGE and Western blot showed that the recombinant protein was successfully induced to express under 0.2 mmol·L-1 of IPTG at 11oC for 8h. Recombinant His-BnALS3 (about 67 kD) could be detected in both lysate supernate and precipitates. Enzyme activity assay indicated that under the optimal condition of pH 7.0 and temperature of 37ºC, His-BnALS3 had Km 6.55 mmol·L-1 and Vmax 6.40 μmol·mg-1·h-1 respectively.
HMG (high mobility group) gene family is related to transcription, replication, recombination and DNA repair. Bioinformatics and identification of HMG gene families from rapeseed (Brassica napus L.) and its allied species was focused in this study. Evolution, gene structure, expression and orthologous/paralogous genes were analysed on origin and function of B. napus HMG gene family. 45 HMG family members were identified in B. napus and were divided into 5 groups according to the evolutionary tree and gene structure. Chromosome identification results showed that 18 chromosome among 19 had HMG genes, indicating wide distribution of this family. Respectively 47, 45 and 26 orthologous genes were identified among B. napus and the other 3 species as B. oleracea, B. rapa and A. thaliana. 28 paralogous gene pairs were identified in B. napus, but were fewer in other 3 species, which could be related to the multiple genome duplication in B. napus. Expression of 45 BnHMG genes in leaves and roots revealed different patterns.
A total of 135 pairs of SSR markers covering entire soybean genome were used to scan genome-wide of 232 varieties including 6 natural sub-populations and 1 breeding sub-populations. The genetic diversity and variation in and among sub-population, the source of molecular variance, genetic drift among sub-populations, between genetic distance of sub-populations and geographical coordinates were analyzed. The results showed that all markers were polymorphic and all detected loci were homozygous. The number of alleles in different loci ranged from 2 to 4, averaging 2.66 per locus. All varieties were highly homozygous. The variation of population polymorphic information content ranged from 0.2751 to 0.3165, and the whole population was 0.3208. The variation range of Nei genetic distance within the population was 0.3258-0.3594, and the whole population was 0.3711. The genetic variation of soybean in Sichuan and Chongqing area was relatively lower. The genetic identity among populations were higher (GI ≥ 0.8862) and genetic distances were lower (GD ≤ 0.1208), genetic differences of local varieties were smaller among populations, genetic differences between the cultivars and all local varieties were relatively higher. The average gene differentiation coefficient (Fst) among populations was 0.0722, and the average gene flow (Nm) among populations was 3.214. There was a certain genetic communication among natural populations. The principal coordinate analysis showed that the first, second and third principal factors were able to explain 4.97%, 3.54% and 3.33% of the total variation respectively. Which came from the same natural population were basically clustered in the same region. The cluster analysis of varieties also showed that varieties of the same natural population were not fully clustered in the same genetic group, but had a certain clustering effect. These showed that there was a correlation between the genetic variation of Sichuan and Chongqing soybean varieties and geographical location. The molecular variation analysis of variance showed that 97% of total variation was explained in the population variation, and 3% of total variation was explained among population variation. Mantel convergence analysis showed that there was a significant positive correlation between the genetic distance and geographical location (latitude and altitude) distance of the natural populations of local varieties (R2=0.723). Research showed that the genetic richness of soybean germplasm resources wasn’t high in Sichuan and Chongqing. The current breeding varieties didn’t contain all the genetic variation in the region. We need to further expand and introduce excellent genetic resources to enhance the utilization of local resources. A few improved varieties adapted to high altitude ecotype could be used in breeding by introducing a minority of genes adapted to high altitudes into excellent lines.
In order to elucidate the regulating basis of drought tolerance in peanut, the experiments of drought and re-water after drought were carried out on 10 different peanut accessions, combined with the transcriptome to analysis the regulation mechanism of drought resistance of different peanut lines. Waliyar Tiga， which was collected from Africa，showed the best drought tolerance, followed by kQ044kangqing, Zhonghua 16 and Zaohuasheng. The physiological results showed that there were significant differences in root/shoot ratio between drought-tolerant and drought-sensitive peanuts. Transcriptome analysis demonstrated that differential expressed genes between drought tolerant and susceptible peanut lines were obviously enriched in oxidative phosphorylation, photosynthesis and secondary metabolites pathways; the genes expression of auxin signal pathway in drought-tolerant peanuts lines were significantly lower than that in the susceptible ones. The physiological and transcriptome data demonstrated that enlarged root system, enhanced energetic, secondary metabolism and the inhibition of growth were the four aspects contributing to drought stress tolerance in peanut. Both Zhonghua 16 and Waliyar Tiga have vigorous root and energetic metabolism, and the root\shoot ratio was even larger in Zhonghua 16 than Waliyar Tiga, the reason for the conflict could be caused by the larger leaf area which resulted greater water loss. This study provided a theoretical basis for drought-tolerant peanuts breeding, and provided a way to further study drought tolerance regulation mechanism of peanut.
In this study, the characters of intercropping soybean were identified with an aim to screen high yield soybean germplasms for intercropping pattern. The correlation between yield traits and 11 agronomic traits of 50 soybean materials were analyzed by grey correlation degree analysis, and their adaptability and yield stability analyzed by GGE-biplot method. The results showed that the variation coefficients and the generalized heritability of agronomic and yield traits were 6.87%-77.32% and 0.62-0.97 in two years respectively. Generalized heritability of 9 characteristics was greater than 0.8, such as growth period, vegetative growth stage and hundred-grain weight. The correlation analysis showed that the yield per plant was significantly positively correlated with stem diameter, growth period, plant height, node number, branch number, bottom pod height, vegetative growth, 100-seed weight, pods per plant, seeds number per plant. The grey correlation degree analysis showed that the order of the correlative degree of main characteristics to yield was stem diameter > growth period >vegetative growth > pods per plant > 100-seed weight > bottom pod height > seeds number per plant > branch number > node number on main stem > plant height > reproductive growth period. GGE-biplot method analysis showed that Fendou 79, Zhongzuo J7018 and Wandou 30 were suitable for intercropping in Shandong.
In rapeseed tissue culture, tenuous and short roots lead to low survival rate of transplanted. To modify the rooting system and to ensure related study, shoots regenerated from a semi-winter oilseed rape cultivar transformed with empty pCAMBIA1301 vector were used. Rooting time, root length, shoot length, shoot weight and plantlet biomass were investigated to assess rooting efficiency of basal medium MS and B5 combined with agar, agarose and phytagel as gelling agent. Results showed that, considering the cost, B5 agar medium containing 20 g/L sucrose were the optimal formula. The optimized rooting medium formula could increase root length and number of in vitro plantlets, and thus enhance the survival rate after transplanting.
For scientific and reasonable evaluation of rapeseed (Brassica napus L.) varieties with high yield and broad adaptability, GGE biplot was used to visualize the yielding ability, stability and adaptability of the candidate winter rapeseed lines in two groups (A and B) national winter rapeseed trials during 2016-2017 in the lower reaches in the Yangtze River Valley. Four main results were: 1) The main effects of genotype (G), environment (E) and genotype by environment interaction (GE) were significant (P<0.01) for yield. Among them, E accounted for 66.0% of the treatment total variation in the sum of squares on average, whereas G and GE accounted for 8.1% and 12.6% respectively. 2) New varieties Dehuiyou 88 and Zhongyou 600 had prominent high-yield and excellent stability, while Chuangyou 17 had high yielding potential but poor stability in both groups. Qinyou 10 (CK) was of middle performance with preferably yield and relatively poor stability. 3) The integrative performance (i.e. ideal index) of Zhongyou 600 in combined evaluation of high yielding and stability was significantly superior to other candidates, and Dehuiyou 88 took the second place, while Qinyou 10 was of medium performance. 4) Dehuiyou 88 had the widest dominant suitable planting areas; Chuangyou 17 was also suitable for a larger area, but performed poorly in some regions; Qinyou 29 and Zhongyou 600 showed good adaptability; Qinyou 10, Qinyou 29, Ningza 118, Ningza 158, Ronghuayou 50 and Su 6006 were only suitablefor certain particular regions.
In this study, Ji-yu 86, a soybean variety introduced in the early stage and super high-yielding in Xinjiang, was selected for this research, and Xin-dadou 23 with high yield potential in local area as control. The growth period, agronomic traits, leaf area index, net assimilation amount, leaf productivity and yield components were compared in order to elucidate the dry matter accumulation and yield traits of Jiyu 86 for the super high yield in Xinjiang, and to further provide theoretical basis for the high yield of Jiyu 86, and to assist in breeding super high yield cultivars. The results showed that after the first flowering stage, the root biomass of Ji-yu 86 was significantly higher than that of Xindadou 23, and flower pods biomass of Jiyu 86 was also relatively higher than that of Xindadou 23 in the whole growth period. Moreover, the pod dry weight of Ji-yu 86 was 16.7% higher than that of Xindadou 23 at maturity stage. Leaf area index and net assimilation of both cultivars were peaked at drumming stage, at which time the leaf area index of Xindadou 23 was 80% higher than that of Ji-yu 86. After drumming stage, net assimilation and leaf productivity of Jiyu 86 were significantly (P <0.05) higher than that of Xindadou 23. Jiyu 86 has relatively compact plant structure, suitable plant height, stem nodes number, less branches and significant population heterosis. In addition, single seed weight, 100-seed weight, harvested plant number of Jiyu 86 were significantly (P <0.05) higher than those of Xindadou 23. In general, the plant structure of Ji-yu 86 was reasonable with obvious population advantages, suitable leaf area index and high utilization rate of photosynthetic products, which could assist the transfer of dry matter accumulation from vegetative organs to grain and achieve a super high yield.
In order to explore effects of seed maturity on black peanut growth, this experiment was conducted by using of black peanut variety (Qianhuasheng 7) to study the changes of seed germination, seedling emergence, photosynthetic characteristics and yield of black peanut under four seed maturity by combining indoor and field experiments. The results showed that the light assimilation ability and photosynthetic rate of seedling using more mature (T2) and mature (T3) peanut as seed were higher than using the immature (T1) peanut kernel as seed. The germination potential, germination rate and emergence rate of peanut increased first and then decreased with seed maturity, and the highest value was treated at T3, and the value of germination potential, germination rate and emergence rate of treatment T2, T3, T4 were significantly higher than those of T1. In addition to the number of bearing branches, the yield and agronomic traits of peanut increased first and then decreased with seed maturity, and pod yield of T3 treatment was significantly higher than that of other treatments. Therefore, mature black peanut was selected as seed, which had good seedling emergence, strong light assimilation ability, high net photosynthetic rate and high pod yield, and was suitable for seed.
Seed-flooding is one of the most important adversity stress in south summer soybean production in China. For researching the seed-flooding tolerance difference of soybean germplasms, 137 materials were treated for 120 hours simulating the field seed-flooding condition. The electrical conductivity of soybean soaking liquid was determined. Germination potential percentage (GPP), germination percentage (GP), normal seeding percentage (NSP), slightly injured seedling percentage (SISP), injured badly seedling percentage (IDSP) was counted through standard germination test. Multivariate statistical analysis, such as principal component analysis and cluster analysis, were used. Comprehensive and sample seed-flooding tolerance index were used to classify the soybean germplasms. Results showed that the CV of electrical conductivity, GPP, GP, NSP, SISP, IBSP et al. was more than 50%. 100 seeds weight had significant negative correlation with GPP, GP and NSP, and significant positive correlation with electrical conductivity. Two principal components ?healthy factor? and ?transition factor? were extracted, with contribution ratio 64.45% and 15.83% respectively. Based on two contribution ratio matrix, 137 soybean materials were clustered into 3 types. And 5 types of these materials were clustered by comprehensive index method，9 soybean materials showed high seed-flooding tolerance. 5 soybean materials showed both high seed-flooding tolerance in comprehensive index and sample index method. 5 soybean materials could be used in seed-flooding tolerance selection and identification work. Electrical conductivity of soaking liquid together with germination potential percentage could be used as the selection index in the soybean mass seed- flooding tolerance selection.
To screen the drought-tolerant Carthamus tinctorius L. genotypes growing in Sichuan Province of China, the influences of drought stress on photosynthetic characteristics, contents of osmoregulation substance, protective enzyme activities and the other physiological characteristics of two drought-tolerant and two drought-sensitive varieties were studied at safflower seedling stage by drought stress. The results showed that the net photosynthetic rate, stomatal conductance, intercellular CO2 concentration and transpiration of 4 safflower varieties declined under the drought stress, and the difference among the different varieties was significant (P﹤0.05). The decrease of photosynthetic characteristics of drought-tolerant varieties (lines) was lower than that of the drought-sensitive varieties (lines). The contents of permeability regulation substance of leaves such as soluble sugar, soluble protein, proline and the protective enzyme activities of SOD, POD and CAT were all increased in 4 varieties (lines) under drought stress. Moreover, the increase rate of drought-tolerant varieties was higher than that of drought-sensitive. The increase rate of MDA content in drought -tolerant varieties was also lower than that in drought-sensitive varieties. Two principal components were obtained by principal component analysis of 11 indexes, and the cumulative contribution rate was 89.50%. The net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, transpiration rate, SOD and CAT activities were used as the primary principal components of drought resistance index, while soluble protein and proline were used as the secondary principal component of drought resistance identification index.
Field split plot experiments were carried out to explore the effect of interaction between Zinc fertilize and shade on development, disease resistance and yield of peanut. The results showed that application of zinc fertilizer improved zinc content in different parts of peanut, leaf SPAD value and content of soluble sugar, protein and auxin in leaf, meanwhile reduced disease rate of peanut with the reduction rate of 7.1%, contributed to the increase of peanut yield by 19.4%, compared with no zinc fertilizer. In the same application rate of zinc, however, zinc content in different parts of peanut and leaf SPAD values in different growth stages decreased with the increase of shade degree, content of soluble sugar, protein and auxin in leaf increased, incidence of peanut disease average increased by 4.8 and 10.2%, pod yield of peanut average decreased by 16.5% and 10.0% with the shade degree increased. Under 30%, 70% shade degree, pod yield of peanut with applied zinc fertilizer increased by 21.1%, 25.0% respectively, compared with no zinc fertilizer. In our experiment, 30kg/hm2 of zinc fertilizer was applied, which made the peanut more resistant to low-temperature and less-light, anti-disease performance, increased pod yield significantly, that could be widely used in peanut production.
In order to explore the molecular mechanism of sunflower resistance to Sclerotinia sclerotiorum, eight defense related genes and the key regulatory genes in SA, JA and JA/ET signal pathway were screened on the basis of the differential expression gene analysis of the transcriptome of Helianthus annuus induced by S. sclerotiorum. qRT-PCR results showed that the genes expression of sunflower defense enzymes had greater increased than that of control when sunflower infected by S.sclerotiorum. The expression of SOD, POD and GST at 6h was 25.02, 22.34 and 26.25 times higher respectively than that of control. The expression of PAL, CAT and CHS at 12h was 23.89, 24.23 and 24.89 times higher respectively than that of control. The expression of GLU and CHI were up-regulated first and then down-regulated, GLU was 23.66 times higher at 24h, and CHI was 22.13 times higher at 36h than that of control. At the same time, the transcriptional levels of regulation genes in signal pathways of sunflower SA, JA and JA/ET had greater variation than that of control. The expression of PDF1.2 gene in JA/ET pathway and NPR1, MPK4 and EDS1 in SA-JA pathway was sharply up-regulated, which showed that many kinds of resistance signal transduction pathways and defense responses of sunflower were induced by S. sclerotiorum, suggesting that the molecular mechanism of sunflower infection on S.sclerotiorum was regulated by multiple gene network system.
In order to further analyze soybean structure mechanism of resistance to soybean pod borer Leguminivora glycinivorella and define its quantitative indicators, 160 spring soybean materials from northeast China were selected to study the characteristics of pod surface structure, forty-one of gradient varieties with single factor structure characteristics were screened. The investigation of oviposition selecting of Leguminivora glycinivorella to soybean varieties with different pod hair and the newly hatched larvae boring in different varieties of pod shell were carried out respectively. The results showed that the frequency distributions of pod hair density, pod hair length, cuticular layer, subcutaneous thick cell layer, middle epidermis cell layer, inner wall cell layer thicknessof 160 breeds showed certain regularities, which can be represented by equation models. Pod hair density had significant positive correlation to single pod egg deposition number. The greater the pod hair density, the more egg of Leguminivora glycinivorella on the pods, which was consistent with the Logistic equation ySingle pod egg loss=6.22/(1+exp(3.17-0.88x)). Significantly negative correlation existed between pod hair length and single-pod egg amount, which was consistent with the quadratic equation ySingle pod egg loss=-23.98+27.82x-6.89x2. When the length of pod hair was more than 2.00mm, the amount of eggs decreased with the increase of the length of pod hair. There was no significant correlation between the thickness of epidermal cuticle and the thickness of the subcutaneous thick-walled cell layer and the pod rate of newly hatched larvae. Though the thickness of mesocarp cell layer and endothecium cell layer was negatively correlated with the boring rate of newly hatched larvae and the exponential function curve equation fitted zMedium peel==142.94exp(-0.70x), Logistic equation zInner wall=103.64/(1+exp(-5.28+20.29x)), respectively. It showed that the thicker the mesocarp cell and endothecium cell layer, the stronger resistance to Leguminivora glycinivorella. The results would provide scientific basis for breeding soybean varieties resistant to soybean pod borer and for evaluating insect resistance.
A high-throughput and accurate counting of cysts is an important prerequisite in the study of Soybean cyst nematode (SCN, Heterodera glycines Ichinohe). The fluorescence imaging technology has been used to count the cysts using halogen or Xenon as the excitation light source. However, because of the low energy of excitation light source, this technology only has good counting results on fresh white cysts rather than old brown cysts with weak fluorescence. In this study, we used laser as the excitation light source and explored two pairs of combinations of excitation and emission wavelengths, which were 470nm and 535nm, and 532nm and 620nm, respectively. The results showed that, under these conditions, the brown cysts could also be obtained with high contrast and clear pictures. Combined with the use of automatic counting software, cyst-counting could be conducted in an accurate and high-throughput way.
To analyze the sensitivity of enzyme-linked immunosorbent assay (ELISA) for aflatoxin M1, the differences of hapten coupling ratio -based indirect competitive enzyme-linked immunoassay were compared. The aflatoxin M1 and CMO were used to synthesize the hapten of AFM1, and then coupling with bovine serum albumin (BSA) to generate artificial antigen AFM1-BSA. After identifying the conjugate by means of UV, ic-ELISA and fluorescence spectra analysis, the results showed that the artificial antigen AFM1-BSA was effective. Furthermore, the artificial antigen and commercial antigen coupling ratios were 0.7 and 6.3 respectively. With the same methods, the artificial antigen coupling ratio was only 1/9 as that of commercial antigen, which provided reference for further research and comparison. Two kinds of antigens were analyzed by laboratory-made antibodies 1C11, 2C9, LM13 and LM47 against aflatoxin M1. The results showed that four antibodies had high sensitivity to commercial antigens, and only 2C9 could react with the artificial antigen AFM1 -BSA and the sensitivity was 1.997 ng/mL, which was significantly lower than that of AFM1-BSA as coating antigen (the sensitivity of 0.047 ng/mL). This study revealed that the high coupling ratio antigen could improve the sensitivity of immunoassay for the antibody prepared by the same toxin AFM1. These results provided a scientific basis to further establish a high sensitive and complete enzyme-linked immunosorbent assay system for aflatoxin M1.
To improve the production efficiency and protein content of rapeseed meal, single bin efficient screen was studied with rapeseed prepress-solvent extracted meal as raw material for concentrated rapeseed meal protein preparation. R-R distribution function of particle size distribution and correlation between protein and phytic acid were analyzed. Single well high efficiency flat sieve was used to produce protein concentrate. The effects of feed rate and rotation speed on screening effect were studied by ratio of screened material and protein content under the sieve. From the particle size distribution and composition of different sizes of raw materials, the optimum conditions were found as follows: inlet flow rate was 2 kg/min, rotation speed of the flat screen was 260 r/min. Under these conditions, the obtained protein content was 56.99% with yield of 26.66%. The concentrated protein obtained by alcohol elution could reach 60.155%.
Interspecific hybridization and allopolyploidization lead to the origin of many important crops, and the cultivated Brassica allotetraploids have become one of the most widely used models. During last two decades, it has been popular to study how the allopolyploids regulate and coordinate genetic behavior and gene expression of genomes from different progenitors. By using the constantly developing molecular biological technology, dynamic nature of polyploid genomes has been revealed in Brassica and other allopolyploids, with the occurrence of genetic and epigenetic alterations at the initial stage and during evolution process. The obvious changes have also been found in gene expressions, including non-additive expression, transgressive expression, expression level dominance, homoeolog expression bias, effects of dosage-balance, etc. These remodulations of genome structure, epigenetic and gene expression patterns have contributed to the evolutionary success of novel allopolyploids into new species in nature.
Multilocular rapeseed has great potential to increase rapeseed yield and is an important crop germplasm resource. At present, domestic and foreign scholars have conducted in-depth researches on multilocular rapeseed. In this paper, we reviewed the research progress from the aspects of the species, anatomy, genetics, gene mapping and cloning of multilocular rapeseed, and pointed out the existing problems and prospects for the study on the multilocular trait of rapeseed, which could provide the theoretical basis for the utilization of multilocular trait in rapeseed.
With the development of biotechnology, there will be more and more researches on genetic improvement of peanuts through genetic engineering. As an important molecular tool, promoter can drive exogenous elite gene expression in peanut, and its identification has important application value in molecular breeding of peanut. In order to facilitate primary peanut researchers to identify promoters, we gave a brief introduction for the structure characteristics and research methods of plant promoters. Meanwhile, research progress of peanut promoter are reviewed, and the future research focus of peanut promoters and the application prospect of peanut promoter was prospected, which provided references for the identification and application of peanut promoter.
