CRISPR (clustered regularly interspaced palindromic repeats)/Cas9 (CRISPR-associated 9) system is the simplest and most commonly used targeted genome editing tool, which has shown the enormous application potential in many fields. The mutation efficiency mediated by CRISPR/Cas9 was affected by many factors, it used transient expression technique to evaluate the mutation efficiency of Cas9/sgRNA vector before generating the mutant plants, which was a favourite way to improve research efficiency. In this study, we developed an Agrobacterium-mediated transient expression technique, Wichita was easy to maneuve and cost-effective for rapid testing the efficiency of CRISPR/Cas9-mediated genome editing. Cas9/sgRNA-Tgt1/2 construction targeted BnaC.WRKY11.a was constructed to mediate multiple mutations with Tgt1 (Target1) and Tgt2 (Target2) as targets in B. napus. Multiple genome modifications were achieved in B. napus after agroinfiltration by Cas9/sgRNA-Tgt1/2 construction. The frequencies of targeted mutagenesis mediated by Tgt1 and Tgt2 were in the range of 11.2%-82.2%. Beyond that, Tgt3 (Target3) targeting to BolPDS3 was designed, Cas9/sgRNA-Tgt3 construction was generated for genome editing in B. oleracea. Transient mutation experiments showed that mutations happened in BolPDS3 at Tgt3. Most of the mutations were deletion mutations, of a range between 1-18 bp. A few of the mutations were insertion and substitution mutations. Our results showed that vacuum-assisted infiltration by Agrobacterium could be an effective transient expression method being cost-effective with easy maneuverability for B. oleracea and B. napus, it could be used for rapid detection of the mutation efficiency mediated by CRISPR/Cas9.
Synthetic B. napus is an important type of materials for genomic research and germplasm improvement. But there are abnormalities in the process of meiosis which lead to genetic instability and pollen fertility decline in synthesized B. napus. For better understanding molecular mechanism of meiosis, sequence of DMC1 homologous, the important plants meiotic gene was isolated from B. napus. Primers were designed based on conserved cDNA sequences from homologous genes, and the DMC1 sequence on A01 chromosome was isolated from B. napus. The amino acid sequence alignment showed that BnDMC1.A01 encoded protein containng typical conserved domains of DMC1 had also been reported in rice and maize. At the same time, 6 haplotypes of the gene were obtained by sequencing analysis of BnDMC1.A01 in 19 accessions of B. napus; 2 SNPs resulted in mutation of amino acid residues were found and they distributed in all 3 types of B. napus. Gene expression showed that BnDMC1.A01 expression level in synthesized B. napus was higher than that in traditional B. napus. However, whether these variations in amino acid or gene expression cause abnormal meiosis in synthesized B. napus still need further elucidation.
In order to improve the breeding efficiency and predictability on hybrid soybean, and finally realize the the aim of higher soybean yields, 5 soybean cytoplasmic male sterile lines as the mother lines and 12 restoration lines as the father lines were used to developed 60 combinations according to NCII method. Combining ability of yield-related traits, heterosis of yields, pods and seeds per plant, and seeds per pod were evaluated. Among the hybridized combinations, the positive mid-parent yield heterosis combinations accounted for 55%, heterobeltiosis combinations accounted for 30%, and more than 20% of mid-parent and over-high parent heterosis accounted for 15%. The mid-parent heterosis of pods and seeds per plant, and seeds per pod accounted for 55%, 56.67% and 23.33% respectively, and the over-high parent heterosis rate was negative. The yield of general combining ability （GCA） of 17 parent materials varied from -27.94%~56.62%，only 6 parental lines showed a positive effect. In addition， Special combining ability （SCA） of 30 combinations all showed a positive effect. Heterosis of yields and seeds per plant were related to SCA; GCA was significantly correlated to yield traits. The seed number of mid-parent heterosis was significantly related to the pod number of mid-parent heterosis， the heterosis yields of over-high parent heterosis， pods and seeds per plant， seeds per pod. SCA had no correlation with GCA in the 4 yield-related traits. The higher yield heterosis combination parents had a higher general combining ability and a higher special combining ability.
In this paper, the evolution of main agronomic traits, yield and quality of 84 soybean varieties released in Shanxi Province from 1973 to 2017 were analyzed. The results showed that the growth period, plant height and main stem, reached the highest in 1980s and then gradually reduced, but slightly increased after 2010. The pod number reached the highest in 1990s. As the years progress, the yields had increased with the continued increase of 100-seed weight, although simultaneously the number of branches and pods per plant declined. The yield increased from 1 992.5 kg/hm2 in 1970s to 2 964.34 kg/hm2 in 2010s with the increase ratio of 48.8%. The content of fat and oil was the highest in 2010s, mainly due to the increase of protein content. At the present, the majority of soybean varieties in Shanxi Province are semideterminate varieties, meanwhile determinate soybean varieties proportion continues to increase and indeterminate soybean varieties proportion is declining, which occupied the largest percentage in 1990s. The proportionof with the round and oval leaves has declined, while the proportion with Phi coniferous and oval leaves has increased, which are mainly resulted from the more efficient use of light energy.
Sunflower (Helianthus annuus L.) is an important oil crop. The achene oil is wrapped with oleic acid, linoleic acid and unsaturated fatty acids. Sunflower oil is considered a high quality edible oil and is primarily used as such. However the oleic acid formation mechanism have not been well characterized. In this study, two sunflower genotypes, previously classified as higher oleic acid concentrations resource (J9) and lower oleic acid concentrations resource (NK244), were used as research object. We used RNA-Seq to discuss and compare the transcriptome profile of the achene of 20 days after pollination. After the multiple comparisons, 5447 DETs (differentially expressed genes) were observed in these two genotypes, including 2709 up-regulated genes and 2738 down-regulated genes. DETs were further researched by bioinformatics analysis, and 2218 DETs were mapped into 3 GO categories, including 57 subcategories. Most of the up-regulated genes were demonstrated to participate in catalytic activity, heterogeneous enzymes, carbohydrates binding, DNA binding, lipid bing, carbohydrate process, lipid metabolism, fatty acid biosynthesis. Most of the up-regulated genes were demonstrated to involved in protein kinase activity, ATP bing, protein phosphorylation, tRNA ammonia acylation of protein translation, cellulose biosynthesis. KEGG analyses showed that 799 DEGs were classified into 28 pathways, and carbohydrate metabolism and lipid metabolism pathways included more DEGs. These finding could assist a better understanding of oleic acid formation mechanism of sunflower for quality breeding and germplasm innovation.
In order to obtain information of phospholipases in Camelina Sativa, transcriptome profiles of embryos at two developmental stages of 10 days (DAF10) and 20 days (DAF20) after flowering were analyzed by 454 pyrosequencing. A total of 521 507 and 310 125 clean reads were produced in DAF10 and DAF20, respectively. The distribution of clean reads length was mainly 341 bp-560 bp. 25 398 and 23 678 unigenes were obtained with the average length of 630 bp and 654 bp in DAF10 and DAF20 by de novo assembly, respectively. On this basis, we screened all unigenes of these 2 samples and obtained 36 candidate phospholipase genes. 36 phospholipase genes were divided into 4 classes, and 4 were phospholipase A1, 9 were phospholipase A2, 10 were phospholipase C, and 13 were phospholipase D. The phospholipase gene family mainly involved in glycerophospholipid metabolism, ether lipid metabolism and inositol phosphate metabolism pathway, as shown in Pathway analysis. In details, all 4 PLA1 genes did not participate in any metabolism pathway, and only Unigene19110, one of PLA2 nine members, played an important function in glycerophospholipid metabolism and ether lipid metabolism. PLC genes, Unigene443, Unigene8071, Unigene8213 and Unigene7311, not only took part in glycerophospholipid metabolism and ether lipid metabolism, but also in inositol phosphate metabolism. Among 13 PLD members, 10 of them except Unigene18630, Unigene17966 and Unigene441 were all involved in glycerophospholipid metabolism and ether lipid metabolism pathway.
Suaeda salsa（Suaeda salsa L.） seeds have high oil content and rich unsaturated fatty acid composition, so they have great potential for development and utilization as special oil crop. In this study, the Illumina HiSeq 2000 high-throughput sequencing platform was used to detect and analyze the transcriptome of Suaeda salsa. 106647 Unigenes were produced, and 76755 of them were functionally annotated by blasting the online protein databases. In order to comprehensively study the classification and expression pattern of genes related to lipid synthesis in Suaeda salsa, the key genes involved in lipid synthesis in seed and leaf transcriptomes of Suaeda salsa were screened and their differential expression profiles were comparatively analyzed. A total of 45 Unigenes encoding key enzymes involved in fatty acid (FA) biosynthesis were obtained, of which 25 Unigenes were up-regulated in seeds of developing stage. In comparison to leaves, a number of Unigenes encoding ACCase, KAS III and EAR, and 5 out of 7 Unigenes encoding acyl carrier protein desaturase (SAD) were significantly up-regulated in developing seeds, which might be related to the formation of unsaturated FA in Suaeda salsa seeds oil. A total of 30 Unigenes encoding key enzymes involved in the synthesis of triacylglycerols (TAGs). Among them, 16 Unigenes were up-regulated in developing seeds, and multiple Unigenes encoding GPAT and DGAT were significantly upregulated. This study could make great contribution to the analysis of the regulation of lipid metabolism pathways and the exploration of the key genes involved in the lipid synthesis of Suaeda salsa.
To explore the interaction mechanism between substrate and phospholipid: diacylglycerol acyltransferases (PDATs) in Arabidopsis thaliana and Saccharomyces cerevisiae, phylogenetic relationships of PDAT in plants, fungi, and other species were investigated by bioinformatics approaches including three-dimensional structure simulation and molecular docking. The binding model between PDAT and specific phospholipid was created by molecular docking. The key amino acids of intermolecular interactions between phospholipid substrate and PDAT were then predicted. Amino acids including W153，E310，D313，V627 in protein sequences of AtPDAT might have played an important role in substrate-binding domain on the base of molecular docking between several phospholipids of AtPDAT and sequence alignment of PDAT proteins. This result accurately revealed the binding site of substrate to PDAT as well as the catalytic domain of candidate PDATs, and provided guidance for future improvement of PDATs.
To explore the effects of seed treatment with Gibberellins (GAs) and uniconazole on emergence of seedling and strong seedlings, the stalk traits, the length of node, the petiole, the leaf photosynthetic characteristics and the yield of soybean were measured after GA3 and uniconazole treatment by seed soaking. The results showed that regulation of GA3 and uniconazole on plant height had varied effectiveness with different application time, and it was more effetive in the seedling stage. GA3 increased the plant height, and uniconazole decreased the plant height. In the full flowering, podding, and filling stage, the plant height did not show significant difference between the treatment and the control group. GA3 treatment significantly decreased the plant height for the 1st, 3rd, 10th, 12th, 13th, 14th shorter internode in the harvesting stage. Uniconazole treatments decreased the lengths of the 1st internode, but did not affect the height of plant. GA3 and uniconazole treatments could have contributed to the lodging resistance by producing thicker and stronger stem. GA3 treatment decreased the petiole length on 8th -10th node, and increased the petiole length on the 14th-18th node, and uniconazole treatment could increase the petiole length on the 7th-12th node. Two growth regulators changed the canopy structure by regulating the node length and the petiole. The petiole length change was correlated with the change of its contribution rate to yield in the same node, and the correlation coefficient were 0.622 and 0.585, P<0.05. Uniconazole treatments significantly increased the chlorophyll content at 7 d, 14 d, and increased transpiration rate (Tr) at 14 d, and significantly increased the grain number per plant, the pods number per plant and yield. The mean production in these two years increased 15.5%. These results indicated that uniconazole treatments affected the soybean yield by changing the canopy structure and the photosynthetic characteristics.
Due to the higher supply of fertilizer and water, and the larger planting density, the high yield peanut is prone to excessive growth and leaf diseases exacerbation at middle growth stage, and easily premature senility caused by absence of fertilizer at late growth period. To solve this problem, our research group set up the ?three prevention and three promotion? regulation techniques: Precise chemical control to prevent plant lodging and promote material distribution and transportion; Early spraying fungicides to prevent disease and preserve leaf and promote the accumulation of photosynthate; Spraying foliar fertilizer to prevent premature senescence and promote full pods. Under high yield cultivation conditions, 4 treatments including CK (ordinary managements), T1 (precise chemical control), T2 (precise chemical control and early spraying fungicides) and T3 (precise chemical control, early spraying fungicides and spraying foliar fertilizer) were set up to further verify the effects of ?three prevention and three promotion? regulation techniques. The results showed T1, T2 and T3 treatments significantly improved SOD, POD, CAT activities and the chlorophyll content of the leaves and the root activity, meanwhile reduced MDA content at pod filling stage and maturity stage of high yield peanut, and the most significant effect was T3 treatment, followed by T2 treatment. T1, T2 and T3 significantly reduced the stem height, lateral branch length and node number of main stem, and increased the branch number, the leaf number of main stem and leaf area index at maturity stage. To regulate the plant growth above the ground part was the main effect of precise chemical control, while hold the larger leaf area index in the later growth stage was mainly the effect of early spraying fungicides and spraying foliar fertilizer. T1, T2 and T3 significantly increased pod number, pod weight per plant, pod yield per unit area and economic coefficient. The pod yield of T1 was 20.35% more than that of CK, while T2 was 26.39% and T3 was 30.62% more than those of CK. The kernel rate of T2 and T3 increased by 1.84 and 3.02 percentage points respectively than those of CK, indicating that T2 and T3 treatments could improve seed plumpness. The application of ?three prevention and three promotion? regulation techniques will be one of the key technical measures to further increase pod yield under high-yield cultivation conditions.
It is a critical step to transplant regenerated plantlet derived from wild species or genetic transformation after tissue culture in peanut. Low survival rate and weak growth vigor of transplanted seedlings in the field has long been an obstacle. To solve this problem the grafting technology of peanut was applied and optimized in the present study. The seedlings of black-seed variety-Yuhua 0215 cultivated in full dark or full light conditions for 7 days were used as rootstocks. The sterile tissue cultured seedlings of cultivated peanut, wild species, and double diploids materials were used as scions respectively. Using the splicing method, scions were grafted onto the hypocotyls sites of the rootstocks. Our results showed that the survival rate of grafts had significant difference between the two rootstocks. The average survival rate was above 96% in the rootstock that was cultured in full dark condition with the hypocotyls elongated beyond 6cm in length. Moreover, graft survival rate significantly varied among scions of different peanut varieties and different types. The graft survival rate of cultivars was higher than that of wild species, and the survival rate reached 100% when grafted onto rootstocks cultured in full dark condition. The strategy of using Yuhua 0215 cultured in full dark condition as rootstock greatly improved graft survival rate and graft efficiency. The method also successfully overcame the low survival rate, weak vigor and the rooting difficulty of transplants derived from tissue culture of the wild species, double diploid and transgenic plants, thus had great potential for further applications..
This paper presented effects of four kinds of boron (B) fertilizers, borax (B，Na2B4O7?10H2O)，Ulexite granular (UB, NaCaB5O9·8H2O)，Etibor-48 (EB, Na2B4O7·5H2O) and Colemanite (CB, Ca2B6O11·5H2O) on yield of peanut (Arachis hypogaea L.) and their residual effectiveness on seed yield for rotated oilseed rape (Brassica napus) by field trials. Results showed that basal application of B, UB, EB and CB (B 1.70 kg/hm2) increased yield of peanut and oilseed rape in different extent; increasing rate of yield of each rotated crops of double boron application treatments (B 3.40 kg/hm2) decreased inordinately comparing with that of treatments at normal B application rate. The increasing rates of yield of peanut and oilseed rape did not increase with the increasing of B dosage when the hot water soluble boron (HWSB) content of soil was 0.49 mg/kg. The HWSB content of all the B application treatments were higher than 0.5 mg/kg when crop were harvested. The boron content of straw and crop yield were also higher than that of no boron treatment. Basal application of these boron fertilizers could significantly increase the HWSB content. UB and CB, which had a more significant increasing rate in Crop2 and Crop3, could be used as basal fertilizers according to their great slow-release properties and soil available B content in agricultural production.
To explore cultivation technology for high-yielding and high fertilizer-nitrogen (N) use efficiency for peanuts in shajiang black soil area, a field experiments was conducted to study the effects of N fertilizer management and rhizobia inoculation methods on peanut growth, N uptake and yield. The results indicated that N application increased SPAD value of peanut leaves, effectively promoted the growth of peanuts, and significantly increased the N use efficiency and yield of pods. Under the same rhizobium methods (rhizobia seed dressing or applying to soil), the first branch length, number of total branch, number and weight of full fruit per plant, 100-pod weight, nitrogen use efficiency and yield were recorded at 50% N as basal fertilizer and 50% N top dressing at flowering stage, and 100% N as basal fertilizer treatment. The efficiency of treatments were significantly higher than those of 50% N top dressing at flowering stage and 50% N top dressing at pod setting stage, indicating that high-yielding and high N efficiency of peanuts required a certain N fertilizer rate in the early growth stage. Compared with two inoculation methods of rhizobia seed dressing and N application to soil, rhizobia seed dressing with N fertilizer had a better effect on peanut yield, but no significant difference between two inoculation methods. In the shajiang black soil area of southern Henan province, the 50% N as basal fertilizer combined with a 50% N fertilizer top dressing at flowering stage with rhizobium seed dressing was a better methods for peanut production.
In this paper, a kind of sucrose ester phospholipid complex was prepared with sucrose fatty acid triester (STE) and phosphatidylcholine (PC) as raw materials. And the sucrose ester phospholipid complex was characterized by fourier infrared spectrum analysis (FT-IR), thermo gravimetric analysis (TGA) and differential scanning calorimetry analysis (DSC). Then the application of STE in rapeseed oil dephosphorization process was studied. The results showed that in anhydrous ethanol system, STE and PC generated a new substance----STE-PC complex, which had lower phase change temperature than STE and PC. In addition, STE had some adsorption effects on phosphatide of the rapeseed oils. The dephosphorization rate was 37.2% when the dephosphorization temperature was 55oC, the STE adding amount was 3.5% and the dephosphorization time was 30min.
