Table of Content

16 February 2015, Volume 48 Issue 4

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Genetic Analysis and Mapping of the Rice Male Sterile Mutant oss125

ZHANG Wen-hui, YAN Wei, CHEN Zhu-feng, XIE Gang, LU Jia-wei, LIU Dong-feng, TANG Xiao-yan

Scientia Agricultura Sinica. 2015, 48(4):  621-629.  doi:10.3864/j.issn.0578-1752.2015.04.01

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【Objective】This study is to conduct genetic analysis on the rice male sterility mutant oss125 and clone the mutant gene using modified MutMap method, laying a foundation for further study of the gene function and its application in agricultural production. 【Method】 A male sterility mutant, designated as oss125, was isolated from a mutant library of the indica rice Huanghuazhan (HHZ) generated with ethyl methane sulfonate (EMS) treatment. Phenotype of the oss125 mutant was observed. The oss125 mutant was crossed with the wild-type HHZ, fertility of the F₁ DNA of 30 F₂plants showing the male sterile phenotype were isolated and bulked in equal amount. Bulked DNA was subjected to whole-genome re-sequencing on Hiseq 2000 platform. A modified MutMap method was utilized to identify genomic regions harboring the causal mutation. Linkage of candidate genes with the mutant phenotype was validated with HRM analysis. Sequence analysis of the candidate gene and the expression of the candidate gene in various rice tissues were studied using real-time PCR. 【Result】 Compared with the wild type HHZ, oss125 mutant had normal vegetative growth, inflorescence and flower morphology. 1% I₂-KI staining revealed 85% abnormal pollen and 15% pollen of normal phenotype. But the plant displayed complete male sterility. All F₁ plants derived from the cross of oss125 mutant with the wild-type HHZ were fertile, and the F₂ plants displayed 3:1 segregation of fertile to male sterile plants, indicating that oss125 is controlled by a single recessive gene. Using a modified MutMap method, four candidate SNPs were identified, three in intergenic region, and one in the second exon of OsRPA1a. A single nucleotide mutation (A663C) occured in the coding region of the OsRPA1a gene, resulting in a nonsynonymous mutation (Q221P) in protein sequence. Linkage of the mutation with the male sterile phenotype was validated using HRM analysis. 【Conclusion】 Mutation of the OsRPA1a gene is responsible for the oss125 mutant phenotype. A663C mutation causes abnormal pollen development, leading to complete male sterility. OsRPA1a is essential for meiosis and DNA repair in somatic cells. It was previously reported that a T-DNA insertion mutant of OsRPA1a was reduced in male fertility but completely female sterile. However, oss125 mutant is completely male sterile, and the female fertility appears not affected, suggesting that OsRPA1a involves distinct regions in controlling male and female gametophyte development. The mutation in the oss125 mutant may be in the region governing male gametophyte development but not affecting the function of the female gametophyte development.and F2 plants was investigated.

Identification of Drought Stress-Responsive Genes in Leaves of Brassica napus by RNA Sequencing

LU Kun, ZHANG Lin, QU Cun-ming, LIANG Ying, TANG Zhang-lin, LI Jia-na

Scientia Agricultura Sinica. 2015, 48(4):  630-645.  doi:10.3864/j.issn.0578-1752.2015.04.02

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【Objective】 To identify the candidate genes involved in drought stress response in leaves of Brassica napus thereby exploring the molecular mechanism of drought stress adaptation of it, the transcriptomes of B. napus seedlings leaves under two different conditions were compared using RNA sequencing (RNA-Seq). 【Method】 Total RNA were extracted from leaves of B. napus cultivar ZY821 at six-leaf stage under normal (ZY) and natural water loss (ZY8D) conditions, and then were used for RNA-Seq analysis on the Illumina Hiseq 2000 platform. Ambiguous reads and low-quality reads were filtered using NGSQCTookit v2.3.3. The TopHat2-Cufflinks-Cuffmerge-Cuffdiff standard pipeline was applied to identify the differentially expressed genes (DEGs), taking the B. rapa chromosome v1.5 and B. oleracea Scaffold v1.0 as reference. In order to perform GO term and KEGG enrichment analyses, the up- and down-regulated DEGs were further analyzed using the BiNGO plugin in Cytoscape v3.1.0 and KOBAS2.0, respectively. Subsequently, the qRT-PCR assays were implemented to verify the expression patterns of three representatives of the up- and down-regulated DEGs, respectively. 【Result】 After filtration, a total of 26192312 and 28378899 high-quality reads were acquired in ZY and ZY8D for screening DEGs, 86.6% and 85.8% of the filtered reads derived from ZY and ZY8D could be accurately mapped to the reference sequence, demonstrating the high confidence of the RNA-Seq and the reference. Of the 3 657 DEGs, 1 431 and 2 226 genes were detected to be up- and down-regulated, respectively. GO enrichment analysis indicated that the up-regulated genes were mainly enriched in response to abiotic stress and chemical stimulus, and 127 and 141 out of these DEGs were involved in response to water deprivation and ABA stimulus, respectively. However, down-regulated DEGs were mainly overrepresented in defense response to plant pathogen, protein kinase activity and response to SA stimulus. KEGG enrichment analysis showed that up-regulated genes were significantly associated with phenylpropanoid and carotenoid biosynthesis pathways, and starch and sucrose metabolism, while the down-regulated DEGs mainly enriched in plant-pathogen interaction and signal transduction pathways of ABA, SA and jasmonic acid (JA). The results of qRT-PCR analysis of six DEGs were consistent with those of RNA-Seq data, further confirming the reliability of RNA-Seq results. 【Conclusion】 In total, 3 657 drought stress-responsive genes were identified using RNA-Seq. GO and KEGG pathway analyses identified the overrepresented molecular function categories and pathways of DEGs.

Advances in Research of Physiological and Molecular Mechanism in Seed Vigor and Germination

LI Zhen-hua, WANG Jian-hua

Scientia Agricultura Sinica. 2015, 48(4):  646-660.  doi:10.3864/j.issn.0578-1752.2015.04.03

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Seed acquired vigor during desiccation stage in seed development, different from the previous opinions, new evidence insisted that seed desiccation is not only involved in water loss process, but also as a significant proportion of the gene expression and metabolic signatures of which resemble those characterized seed germination, implied that the preparation of the seeds for germination began already during seed desiccation. The germination of seeds initiated from water-uptake, accompanied by metabolic recovery, then radicle breaked through endosperm and seed coat and other peripheral completed germination. The main contributor of seed germination success is the quality of the messenger RNAs stored during embryo maturation in the mother plant. In addition, proteostasis and DNA integrity play a major role in the germination phenotype. Plant hormone, as a signal, concentration extremely fewer even approximate to zero, was also important for seed dormancy release and germination. Recently, more and more ideas have considened that the ABA/GA ratio regulats the metabolic transition required for germination. GAs, although required for the completion of germination, are not directly involved in many processes taking place during germination like ABA, which occurred at a stage coinciding with or very close to radicle emergence. It appears that reciprocal downregulation of the respective metabolic pathways accounts for a significant part of GA and ABA interplay, and the α-amylase gene, is transcriptionally regulated by both ABA and GA. In addition to the ABA and GA, recently research found IAA finely regulated radicle emerge that more strictly than cotyledons open during seed germination. Auxin action in seed dormancy and germination requires the auxin response factors 10 and 16 to indirectly control the expression of ABI3. Compared with germination in light condition, seedlings germinated in soil will form a specific organization ‘Apical hook’, its main role was to protect the ‘SAM’. Auxin, accumulating at the concave side, was critical for the formation and maintenance of the hook structure, whereas a release of the auxin maximum correlates with hook opening. To improve the vigor, seed were primed before sowing, the key of which was to control seeds very close to radicle emergence but redried before it in time. In fact, seed stored mRNAs and proteins began to perform its function during priming, and also the molecular biological mechanism was "memorized" at the later redried state, so re-imbibed seeds could quickly germinate. Except as toxic molecules, ROS is also involved in the mobilization of storage and endosperm loose as signaling molecules during seed germination, and it is always interacted with hormone molecule ABA and GA, that also controls seed germination via translation and posttranslational modifications. Methionine metabolism is the metabolism core in seed germination and its metabolites widely regulate the physiological and biochemical reaction of seed germination, such as DNA synthesis, protein stability, chromosome structure formation and remodeling, biotin synthesis, and is also interacted with hormone molecule ABA, GA, ETH, and active oxygen or nitrogen. Recently new evidence insisits on the methionine sulfoxide reductase took part in repair system in plant seed longevity, which also might play a role in seed vigor. This article focused on the domestic and foreign research status on seed vigor, and also provided an outlook of the future research hotspots, such as the senses traits in seeds "preharvest", the molecular mechanism about higher vigor seeds but less field emergence, the important role of auxin in radicle breakout, methionine metabolism, and the methods about seed vigor testing, etc.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY

Effects of Plastic Mulching on Water Consumption Characteristics and Grain Yield of Winter Wheat in Arid Region of Northwest China

YANG Chang-gang, CHAI Shou-xi, CHANG Lei, YANG De-long

Scientia Agricultura Sinica. 2015, 48(4):  661-671.  doi:10.3864/j.issn.0578-1752.2015.04.04

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【Objective】 Strategies and practices are required to increase crop yields and improve water use efficiency (WUE) in arid and semiarid rainfed areas. This study was carried out to determine the effect of different plastic mulching modes on water consumption characteristics, grain yield, and soil water recharge postharvest fallow period for winter wheat in a semiarid rainfed Loess Plateau area of Northwest China.【Method】 This experiment was conducted under arid conditions during wheat growing seasons in 2008-2010 and soil water storage, water consumption amount, grain yield and WUE for winter wheat were analyzed by using four cultivation patterns which were whole field plastic mulching with soil covering on the top of mulch and bunch-seeding (A), whole field plastic mulching without soil cover and bunch-seeding (B), ridges mulched with plastic and row-seeding in the furrow (C), and uncovered and row-seeding (CK), respectively. 【Result】 Results showed that compared to CK, the whole field plastic mulching without soil cover and with bunch-seeding increased winter wheat yield by 64.4%-79.1% and WUE by 22.1%-24.0%, with WUE reached 11.9 to 16.6 kg·hm^-2·mm^-1. The whole field plastic mulching with soil cover on the top of the mulch and bunch-seeding increased winter wheat yield by 43.4%-44.4% and WUE by 8.8%-14.6%, with WUE reached11.0 to 14.8 kg·hm^-2·mm^-1. The ridge-furrow mulching and row-seeding in the furrow increased winter wheat yield by 37.0%-39.3% and WUE by 4.2%-4.4%, with WUE reached 10.0 to 14.2 kg·hm^-2·mm^-1. Plastic film mulching was found to able to increase the soil water storage of 0-200 cm soil profile before the jointing stage, increase water consumption amount from jointing to maturity, and increase the proportion of water consumption after jointing to the total water consumption during the whole growing season. Plastic mulching also improved the use of soil water storage during the whole growth period, enhanced deep soil water usage in arid years, and then enhanced the biomass and grain yield at harvest. Plastic mulching led to a higher soil water consumption over the whole growing season, but the water use efficiency was also increased due to much more increase of grain yield. There was a significant positive correlation between the crop productivity and the soil water consumption during the growth period (r=0.96*). Though the high grain yield of plastic mulching was based on high water consumption, the soil moisture in the 0-200 cm depth with plastic mulching was restored from postharvest to the autumn seeding of the next crop to the level equivalent to the soil moisture in open field. The differences of water consumption among mulching treatments were significantly smaller than those between mulching treatment and open field contrast. By comparison of the three mulching methods, an added feature with the whole field plastic mulching with soil cover on the top of the mulch and bunch-seeding is that the plastic film can be reused for the next crops with simplified cropping operation, helping save film cost, and improving economic benefits, and therefore, it was recommended that is the most appropriate cultivation practice in the experiment. 【Conclusion】 It was concluded that the whole field plastic mulching with soil cover on the top of mulch and bunch-seeding is a highly-efficient cultivation pattern for increasing crop productivity, enhancing water use efficiency, and improving economic benefits for winter wheat in the semiarid rainfed Loess Plateau area of Northwest China.

Effects of Low Nocturnal Temperature on Photosynthetic Apparatus of Winter Rapeseed (Brassica campestris L.)

LIU Zi-gang, SUN Wan-cang, FANG Yan, LI Xue-cai, YANG Ning-ning, WU Jun-yan, ZENG Xiu-cun, WANG Yue

Scientia Agricultura Sinica. 2015, 48(4):  672-682.  doi:10.3864/j.issn.0578-1752.2015.04.05

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【Objective】 The objective of this paper is to investigate the effects of low nocturnal temperature on photosynthetic apparatus of winter rapeseed (Brassica campestris L.) and the associated differences between different cultivars. 【Method】 An artificial climate chamber was used to simulate the effects of low nocturnal temperature on stomatal morphologies, chloroplast ultrastructure, photosynthetic parameters, and dry matter distribution and accumulation in seedling leaves of two winter rapeseed cultivars, Longyou-7 with ultra cold resistance and Tianyou-2 with weak cold resistance. 【Result】 At diurnal/nocturnal temperatures of 20/10℃ (control), the leaves of Louyou-7 had lower growing point than those of Tianyou-2, both showed a creep plant type, a deep green leaf color, and similar levels of leaf chlorophyll content, stomatal conductance (G_s), intercellular CO₂ concentration (C_i), and photosynthetic rate (P_n). After nocturnal temperature drops to 5°C for 7 days, both cultivars had decreased G_s, C_i, and P_n but increased leaf chlorophyll content and root/shoot ratio, indicating that more photosynthetic products were transported with priority to the roots. Tianyou-2 had curved leaf margin and tip with spotty water stains, showed obvious symptoms of cold damage, whereas Longyou-7 had a depended leaf color, a flat leaf shape, and a subsided growing point with no symptoms of cold damage. After treatment at low nocturnal temperature, both cultivars had increased leaf chlorophyll content but the extent of increase differed between Tianyou-2 and Longyou-7 (6.0% vs. 9.6%). During this period, the majority of stomata were closed or semi-closed in Tianyou-2, with higher C_i but lower G_s and P_n than Longyou-7. These observations suggested that at low nocturnal temperature of 5℃, photosynthesis was significantly inhibited in Tianyou-2, mainly because of non-stomatal limitation. On the contrary, most stomata remained open in Longyou-7 at low nocturnal temperature of 5℃, associated with minor decreases in C_i and P_n but no significant differences from data of the control (diurnal/nocturnal temperatures of 20/10℃). These results demonstrated that Longyou-7 maintained relatively high photosynthetic rate at low nocturnal temperature. Regarding chloroplast ultrastructure, the leaves of control plants at diurnal/nocturnal temperatures of 20/10℃ had orderly arranged chloroplasts close to the inner wall of cells, with neat and dense stacks of grana lamellae in an orderly arrangement; the chloroplasts displayed a spindle or single-sided concave shape, containing starch granules which were more and bigger in Longyou-7 than in Tianyou-2. When the diurnal/nocturnal temperatures dropped to 20/-5℃, leaves of Tianyou-2 had the lower part completely dried up and the green-holding leaves with water stains as the symptoms of cold damage, whereas Longyou-7 had interior leaves in a deep green color and the lower part of leaves in a flat shape, with massive red color at the blade surface. With respect to chloroplast ultrastructure, Tianyou-2 had adjacent chloroplasts fused together, with ruptured chloroplast membrane, released inclusions, dissolved grana, and broken stromal lamellae, indicating complete damage of the ordered structure of chloroplast membrane. In contrast, Longyou-7 had chloroplasts retaining complete and clear outer membrane and partial structure of grana, with complete structure of stromal lamellae containing a small amount of starch granules. After treatment at low nocturnal temperature of -5℃, both cultivars had increased C_i and decreased chlorophyll content, G_s and P_n. However, Tianyou-2 had significantly lower chlorophyll content than Longyou-7 and the P_n rapidly declined to 0.210 μmol CO₂·m^-2·s^-1, with 255.2% decrease compared to the control. Longyou-7 had the P_n of 0.434 μmol CO₂·m^-2·s^-1, which was 2.06-fold that of Tianyou-2. These results indicated that Longyou-7 maintained relatively high photosynthetic rate after treatment at low nocturnal temperature. The root/shoot ratio was significantly higher in Longyou-7 than in Tianyou-2, reflecting that the former transported more photosynthetic products with priority to the roots for storage. 【Conclusion】 Low nocturnal temperature caused damage to the structure of photosynthetic membrane of chloroplasts and reduction of P_n in leaves of winter rapeseed, thus influencing photosynthetic processes in this crop. The reduction of P_n was mainly related to stomatal limitation at diurnal/nocturnal temperatures of 20/5℃ and non-stomatal limitation at diurnal/nocturnal temperatures of 20/-5℃.

PLANT PROTECTION

Identification of a Protein both in Tobacco and Rice that Interacts with an HR-elicitor Ssb_X of Xanthomonas oryzae pv. oryzicola

SUN Yu-jing, MA Wen-xiu, CAI Lu-lu, LIU Liang, ZOU Li-fang, CHEN Gong-you

Scientia Agricultura Sinica. 2015, 48(4):  683-694.  doi:10.3864/j.issn.0578-1752.2015.04.06

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【Objective】A highly-conserved single-stranded DNA-binding protein (SsbX) of Xanthomonas oryzae pv. oryzicola, secreted through the type-III secretion system (T3SS), elicits hypersensitive response (HR) in non-host tobacco. The objective of this study is to clarify how it triggers HR. 【Method】 The appropriate period of rice and tobacco leaves were infiltrated with X. oryzae pv. oryzicola RS105 strain to construct rice and tobacco cDNA libraries using Creator^TM SMART^TM cDNA Library Construction Kit, then the qualities of rice and tobacco cDNA libraries were determined by using the special enzyme cutting site SfiⅠof pGADT- SfiAB vector. These cDNA libraries were applied to screen SsbX-interacting proteins on SD/-Ade/-Leu/-Trp/-His medium by yeast two-hybrid system (Y2H). The homology and phylogenetic tree analysis of SsbX-interacting proteins were operated by the method of neighbor-joining using MEGA.4 software. The sublocalization of SsbX with SsbX-interacting proteins in tobacco leaves was observed and imaged under a laser confocal microscope at 488 nm, the excitation wavelength for YFP, and 520 to 550 nm, the emission filter wavelength.【Result】Construction and detection of rice and tobacco cDNA libraries showed the average length of cDNAs inserted into pGADT-SfiAB was 1.0 kb among 20 randomly-selected rice cDNA library clones and 1.2 kb among 20 tobacco cDNA library clones, indicating good qualities of rice and tobacco cDNA libraries could be applied for subsequent research. The Y2H and β-gal assay results demonstrated that SsbX interacted with an actin-depolymerization factor 2 (ADF2) either from rice or tobacco and this couple made AH109 positively grow on SD/-Ade/-Leu/-Trp/-His plate with blue color. Hereby this factor was designated OsADF2 for rice and NbADF2 for tobacco correspondingly. OsADF2 encoded a 139 amino acid (aa) protein, 15.9 kD in molecular weight, and NbADF2 encoded a 137 aa in 15.8 kD. These two proteins were highly conserved with 69.02% identities to each other in protein sequences. Homology and phylogenetic tree analysis displayed that ADF2 proteins in plants were highly conserved in similarity up to 60%. NbADF2 had 69.05% similarities to the homolog of Arabidopsis, higher than orthologs in other dicotyledonous plants. OsADF2 showed 88.81% similarities to the homolog of Setaria italic, higher than orthologs in other monocots plant. Bimolecular fluorescence complementation (BiFC) assay results demonstrated that the presence of OsADF2 or NbADF2 together with SsbX, like a positive control, displayed yellow fluorescence in plant plasma membrane under a laser confocal microscope when the pair proteins were transciently expressed in plant cells mediated by Agrobacterium. 【Conclusion】 The SsbX protein of Xanthomonas, secreted through the T3SS, targets ADF2 in plant plasma membrane to trigger plant immunity. This novel finding provides a scientific clue to further understand how SsbX elicits HR in tobacco plants.

Function Analysis of β₁-tub and β₂-tub in Resistance of Gibberella zeae to Carbendazim

ZENG Fan-song, YIN He-xing, SHI Wen-qi, WANG Hua, YANG Li-jun, GONG Shuang-jun, ZHANG Xue-jiang, XIANG Li-bo, YU Da-zhao

Scientia Agricultura Sinica. 2015, 48(4):  695-704.  doi:10.3864/j.issn.0578-1752.2015.04.07

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【Objective】The objective of this study is to reveal the function of β₁-tub and β₂-tub in resistance of Gibberella zeae to carbendazim.【Method】α-, β₁-, β₂- and γ-tub of 5 carbendazim resistant strains, Js449 (EC₅₀=7.911 μg·mL^-1), Js462 (EC₅₀=6.515 μg·mL^-1), Js484 (EC₅₀=5.031 μg·mL^-1), Js506 (EC₅₀=8.455 μg·mL^-1) and Js519 (EC₅₀=6.280 μg·mL^-1), were cloned and sequenced, and alignment were carried out among these sequences with those of HG-1 (EC₅₀=0.552 μg·mL^-1), a carbendazim sensitive strain, respectively. Relative expression levels of β₁-tub and β₂-tub in Js506, a resistant strain, in response to carbendazim were detected using real-time quantitative PCR (qPCR). The overexpression vectors harboring β₁-tub and β₂-tub were constructed and transferred into HG-1. Hygromycin phosphotransferase gene and flanking sequences of target gene were fused by split PCR. The entire β₂-tub locus was deleted from Js506 and complementation was also performed by protoplast transformation and homologous recombination. Sensitivity to carbendazim, colony growth and pathogenicity of Js506, HG-1 and their mutants were tested. 【Result】  No mutation was detected in α-, β₁- and γ-tub of 5 resistant strains based on DNA sequence alignment with corresponding sequences of HG-1. The multiple sequence alignment for β₂-tub revealed a mutation (Phe 167 Tyr) at the codon 167 in Js449, Js462 and Js506 and at the codon 200 in Js484. A change at the codon 198 from Glu to Gln was also detected in Js519. Expression of β₁-tub was induced in Js506 by treatment of 5 μg·mL^-1 carbendazim at a significant level of 0.05. Carbendazim at 10 μg·mL^-1 did not exert an influence on expression level of b₂-tub in Js506 significantly. Overexpression of β₁-tub in HG-1 resulted in an increase of the value of EC₅₀ up to 2.839 μg·mL^-1 and enhanced resistance to carbendazim (P<0.05). No significant difference was presented in resistance level between the mutant with overexpressed b₂-tub and the wild strain. Two transformants, Δβ₂tub-Js506-1 and Δβ₂tub-Js506-2, were generated by knocking out assay and validation was carried out by hygromycin screening, PCR amplification and Southern blotting. Compared with the parent strain, both two deletion mutants of b₂-tub locus displayed supersensitivity to carbendazim, slower growth of colony, and reduced pathogenicity (P<0.05). Two complementation mutants of △β₂tub-Js506-1, β₂tub-Js506-C1 (EC₅₀=7.521 μg·mL^-1) and β₂tub-Js506-C2 (EC₅₀=7.243 μg·mL^-1), were identified and these biological characteristics were almost restored in the two transformants by genetic complementation. 【Conclusion】Resistance of 5 strains was correlated with point mutations at codons 167, 198 and 200 in the β₂-tub but not with mutations in the β₁-tub sequence. Treatment of culture with carbendazim induced β₁-tub transcript levels and overexpression of β₁-tub improved its resistance to carbendazim. β₂-tub is necessary for resistance of G. zeae to the fungicide, and both two tubulin genes are able to affect the resistance.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Effects of Biochar and Straw Additions on Lime Concretion Black Soil Aggregate Composition and Organic Carbon Distribution

HOU Xiao-na, LI Hui, ZHU Liu-bing, HAN Yan-lai, TANG Zheng, LI Zhong-fang, TAN Jin-fang, ZHANG Shui-qing

Scientia Agricultura Sinica. 2015, 48(4):  705-712.  doi:10.3864/j.issn.0578-1752.2015.04.08

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【Objective】The influences of biochar and straw additions on lime concretion black soil water-stable aggregate composition and organic carbon distribution were studied.【Method】A six-month incubation experiment was conducted here. The experiment included four treatments: CK (no additional materials), B (biochar addition alone), S (straw addition alone) and BS (combined biochar and straw addition). All samples were separated into six aggregate-size classes (>2 mm, 1-2 mm, 0.5-1 mm, 0.25-0.5 mm, 0.053-0.25 mm and ＜0.053 mm) by wet sieving. Organic carbon contents of the aggregates were determined. 【Result】Compared with CK, biochar application did not change aggregate >2 mm fraction, while application of straw significantly increased the amount of >2 mm aggregate. Among all aggregates, application of biochar alone significantly increased the amount of size 0.053-0.25 mm, while straw significantly increased the fraction of 0.5-2 mm by 14%-68%. At the same time, B treatment had no significant influence on mean weight diameter (MWD), geometric mean diameter (GMD) and the contents of macro-aggregates (R_0.25), while MWD, GMD and R_0.25 of S and BS treatments were significantly increased and the fractal dimension (D) of all treatments were extremely lower than CK. In addition, all organic material treatments significantly increased the organic carbon content, among which BS was the highest by 160%, and the content of organic carbon at all levels of aggregate sizes were also highly increased by 54%-353%, as compared with CK treatment. Meanwhile, with the increment of the soil particle-size, it showed the V trend under biochar and the go-up tendency under straw alone on the distribution of organic carbon in aggregates. As for the contributing rates of aggregates to soil organic carbon，the trend of macro-aggregate was S > BS > CK > B, while micro-aggregate had the contrary tendency, and aggregates 0.5-1 mm in size were greater than other aggregate size by 6%-33%. 【Conclusion】 Applying biochar alone could not change the amount of macro-aggregate and its stability. Combined application of straw and biochar significantly increased soil macro-aggregate content and organic carbon content in all sizes of aggregates. In contrast, combined biochar and straw is the best fertilizing measure to improve soil structure and soil organic carbon content in lime concretion black soil.

Effect of Irrigation and Fertilizer Coupling on Greenhouse Tomato Yield, Quality, Water and Nitrogen Utilization Under Fertigation

XING Ying-ying, ZHANG Fu-cang, ZHANG Yan, LI Jing, QIANG Sheng-cai, WU Li-feng

Scientia Agricultura Sinica. 2015, 48(4):  713-726.  doi:10.3864/j.issn.0578-1752.2015.04.09

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【Objective】Water and fertilizer are two major factors for limiting crop yield increase. Unreasonable application of irrigation and nitrogen can hinder yield increase, but increase the soil nitrate accumulation, and reduce the crop quality, water and nitrogen use efficiency. The study was aiming at dealing with the problems of irrigation and fertilization on greenhouse vegetables in semi-arid northwest areas, which through the effects of irrigation and fertilizer coupling on greenhouse tomato yields, quality, and water and nitrogen use efficiency under fertigation. The purpose of this study was to research a high-yield, good-quality and high-efficiency fertigation system for drip-irrigated greenhouse tomato. 【Method】Greenhouse tomato plot experiments were conducted to analyze the responsive rules of irrigation amount and fertilizer rate on tomato growth, yield, quality, distribution of soil NO₃^--N, water and nitrogen absorption and utilization. The experiments consisted of a furrow fertigation treatment (control treatment, 100% ET₀, N240-P₂O₅120-K₂O150 kg·hm^-2), three drip irrigation levels (high irrigation, W₁, 100% ET₀; medium irrigation, W₂, 75% ET₀; low irrigation, W₃, 50% ET₀) and three fertilizer levels (high fertilizer, F₁, N240-P₂O₅120-K₂O150 kg·hm^-2; medium fertilizer, F₂, N180-P₂O₅90-K₂O112.5 kg·hm^-2; low fertilizer, F₃, N120-P₂O₅60-K₂O75 kg·hm^-2), and total of 10 treatments.【Result】The results showed that tomato yield, dry matter accumulation and total nitrogen absorption in drip fertigation treatments were 31.04 t·hm^-2,  3 208 kg·hm^-2 and 73.13 kg·hm^-2 higher than that in the furrow fertigation treatment, increased by 46.9%, 54.0% and 82.4%, respectively. In addition, the fruit Vc content, water use efficiency (WUE) and nitrogen use efficiency (NUE) increased by 61.8%, 46.4% and 76.5%, respectively, but the NO₃^--N content in the soil decreased. In drip fertigation treatments, the highest total dry matter accumulation of W₁F₂ treatment was 9 248 kg·hm^-2, both yield and plant nitrogen uptake were positively related to the amount of irrigation and fertilizer. The fertilization was found to exert more impact on the yield compared with irrigation. The treatment W₁F₂ had thelargest range of yield increase and nitrogen uptake. The WUE decreased and the NUE raised gradually with the increase of irrigation amount and decrease of fertilizer rate. The treatment W₃F₁ produced the largest WUE (47.7 kg·m^-3) and the W₁F₃ treatmentof the largest NUE (65.6 %), respectively. The WUE of W₃F₂ and the NUE of W₁F₂ had much more greater increasing scale than the other treatments. The nitrate nitrogen content in soil was significantly affected by irrigation, fertigation and irrigation-fertigation interactive effects. Nitrate nitrogen content in the soil increased first but then decreased with the increase of irrigation water. Nitrate nitrogen content consistently increased as the increase of fertilizer. Significant nitrate nitrogen accumulation was identified at the lateral boundary of the wetted volume rather than under the dripper. The treatment W₁F₂ with high irrigation water and medium fertilizer produced lower nitrate nitrogen content and more uniform distribution of nitrate nitrogen in the soil than other treatments. Increases in irrigation water significantly reduced fruit Vc, lycopene and soluble sugar contents and their nutrition accumulation, but the quality indicators’ content and nutrition accumulation were increased first then decreased with the increase of fertilizer. The treatment W₃F₂ had the largest Vc and lycopene content as well as nutrition accumulation, and the largest soluble sugar contents as well as larger nutrition accumulation. 【Conclusion】The results indicated that the drip fertigation technology could achieve the goal of high yield, good quality and high efficiency for greenhouse tomatoes. The treatment W₁F₂ with high water and medium fertilizer (100%ET₀, N180-P₂O₅90-K₂O112.5 kg·hm^-2) was preferable for higher yield and NUE interest, as well as lower nitrate nitrogen content in soil; but the treatment W₃F₂ with low water and medium fertilizer (50%ET₀, N180-P₂O₅90-K₂O112.5 kg·hm^-2) was more appropriate in terms of higher WUE and the largest Vc, lycopene and soluble sugar contents.

HORTICULTURE

Toxicity Analysis of Kidney Bean and Construction of Its Prediction Model

LI Jia-nan, YANG Wei, PENG Na, CHEN Chan-you

Scientia Agricultura Sinica. 2015, 48(4):  727-734.  doi:10.3864/j.issn.0578-1752.2015.04.10

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【Objective】 The objective of this study is to analyze the toxicity of varieties of kidney beans in mice cells, and construct a toxicological prediction model of kidney beans, thus providing a basis for breeding low poisonous bean variety. 【Method】The maceration extract of 27 varieties of kidney beans whose content of four anti-nutritional factors had been measured were prepared by methods of homogenization, centrifugation and ultrafiltration. Mouse lymphocyte was isolated by density gradient centrifugation and treated with kidney bean extract. MTS assay was used for detecting the influence ratio of lymphocyte proliferation. A multiple regression equation was generated by SPSS statistical software, combined the content of four anti-nutritional factors with the influence ratio of lymphocyte proliferation, taken influence ratio of lymphocyte proliferation (Y) as dependent variable and the content of four anti-nutritional factors as independent variable. Then, the toxicological prediction model of kidney beans was established by stepwise regression analysis. An acute toxicity experiment was performed on healthy mice in order to verify the feasibility of toxicological prediction model of kidney beans. The mice were separated randomly into three groups. The mice in experimental group were given kidney bean extract by intragastric administration. At the same time, the mice in positive control group were received standard PHA, and the mice in negative control group were treated with only physiological saline. The survival time was checked every day. After death of the subjects, the biopsies of liver, spleen, kidney, stomach and intestine were taken for histological examination under the microscope using HE staining. 【Result】The results of MTS assay showed that the kidney bean extract expressed toxicity to mice lymphocyte, and the toxicity of varieties kidney beans was positively correlated with lectin content. There was a significant difference in the influence ratio of lymphocyte proliferation after treated by kidney bean extract, which was ranging from 13.77% to 85.23% (P＜0.01). A multiple regression equation (Y=-20.88+4.902 X₁+0.258 X₂-3.506 X₃+18.298 X₄) was generated by SPSS statistical software, combined the content of four anti-nutritional factors with the influence ratio of lymphocyte proliferation, taken influence ratio of lymphocyte proliferation (Y) as dependent variable and the content of four anti-nutritional factors (X) as independent variable. Then, the toxicological prediction model (Y=30.837+4.5 X₁) of kidney bean was constructed by stepwise regression analysis. The results of acute toxicity experiments showed that the fatality rate in a week was high in group with high influence ratio of lymphocyte proliferation. The mice that treated with inactivated kidney bean extract by heating could survive more than a week. It indicated that the main toxic substance of kidney bean was protein. The further study of anatomy and pathology analysis displayed that congestive, edema and the inflammatory reaction appeared in the liver, spleen, kidney, stomach, and intestine in experimental group mice, similar to the mice in positive control group. It illustrated that the major endogenous toxin of kidney bean was lectin. 【Conclusion】This study will provide a theoretical basis and technical methods for the genetic improvement of kidney bean quality and utilization.

Identification and Expression Analysis under Abiotic Stresses of the CBL Gene Family in Pear

XU Yuan-yuan, LIN Jing, LI Xiao-gang, CHANG You-hong

Scientia Agricultura Sinica. 2015, 48(4):  735-747.  doi:10.3864/j.issn.0578-1752.2015.04.11

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【Objective】 Calcineurin B-like proteins (CBL) is a kind of Ca²⁺ sensor protein recently found in plants. CBL /CIPK (CBL-interacting protein kinase) signal network system plays an important role in plant stress response such as salinity, drought and chilling. The evolution and structure of Calcineurin B-like proteins (CBLs) in pear were identified and their tissue expression was analyzed for providing a basis for the functions of plant CBL genes and their application. 【Method】 The annotation information of the pear genome was analysed using bioinformatics tools to identify the sequence information of CBL family members in pear. The MEGA 6.0 software was used for multiple sequence alignment, classification, and construction of the phylogenetic tree; per1-based program, GSDS and Clustal X software were adopted to analyze gene structure and conservative. qRT-PCR was further used to analyse the expression of PbCBLs under various abiotic stress conditions. 【Result】 Seven CBL gene family members were successfully identified. Gene structure prediction indicated that PbCBL9 consists of only five introns, while others consist of 7 to 8 introns. The predicted PbCBLs contain four EF-hand domains and a highly conservative number of amino acids between every two adjacent EF-hand domains. According to the phylogenetic analysis, the seven PbCBLs were classified into two subgroups. Differences in gene expression analysis measured by qRT-PCR in leaves of Pyrus betulaefolia Bunge showed that, in NaCl treatment, the PbCBL1 gene expression reached its nadir at 6 h, and then increased after 24 h. On the contrary, the expression of PbCBL2 and PbCBL3 showed a maximum and minimum at 6 h and 24 h, respectively. Meanwhile, PbCBL4 and PbCL8 were significantly increased at 3 h, but then decreased. PbCBL9 expression was clearly enhanced throughout NaCl treatment and reached a maximal expression at 24 h. PbCBL10 exhibited a maximal expression level at 6 h. In the stress condition of 10% PEG6000, the expressions of PbCBL2, PbCBL4, and PbCBL8 all increased, while the expression of PbCBL1 was decreased. PbCBL3, PbCBL9, and PbCBL10 had a minimum gene expression at 6 h, but then increased significantly at 12 h and 24 h as compared with that at 6 h. PbCBL3 and PbCBL10 reached a maximum at 24 h while PbCBL9 reached a maximum at 12 h. Under cold temperature stress (4℃), the expressions of PbCBL2, PbCBL4, and PbCBL8 all increased while PbCBL1 and PbCBL3 both decreased. Fluctuations in PbCBL9 and PbCBL10 gene expression levels were observed, with significant increases around 3 h, followed by a decrease. Under high temperature stress (42℃), the expressions of PbCBL1, PbCBL3, and PbCBL4 all decreased, while PbCBL2 was increased, reaching a maximum at 6 h. PbCBL8, PbCBL9, and PbCBL10 had similar trends overall, with the highest levels at 3 h followed by an obvious decline. Under ABA stress conditions, the expressions of PbCBL3 and PbCBL10 decreased while the expressions of PbCBL2 and PbCBL8 increased. PbCBL1 reached a maximum at 6 h and the expressions of PbCBL4 and PbCBL9 significantly increased at 3 h followed by a decrease, reaching its lowest at 24 h. 【Conclusion】 The pear CBL genome family consists of seven members and among all the seven PbCBLs members, two PbCBLs genes (PbCBL8 and PbCBL9) were induced by salt stress, three PbCBLs genes (PbCBL2, PbCBL4 and PbCBL8) were strongly induced by drought, cold and ABA stress. PbCBL2 may play an important role in pear under heat stress because of its constitutive high level expression.

Analysis of Genetic Diversity and Construction of DNA Fingerprint Database of Xinjiang Apricot Varieties (Lines)

LIU Juan, LIAO Kang, Mansuer·Nasir, SUN Qi, LIU Huan, JIA Yang

Scientia Agricultura Sinica. 2015, 48(4):  748-758.  doi:10.3864/j.issn.0578-1752.2015.04.12

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【Objective】The genetic diversity and relationship of Xinjiang apricot varieties (lines) were studied at molecular level by ISSR, and the DNA fingerprinting database was also established. The result of the study will give the cross breeding, classification and the identification some guidance. 【Method】Six materials with different phenotypes were selected to screen the characteristic primers from 100 ISSR primers which were have clear stripe and main band, rich polymorphism, repeatability, and the varieties with high resolution. The genetic diversity of 48 Xinjiang apricot varieties (lines) were analyzed by POPGENE version 1.32, the number of polymorphism and the percentage of polymorphic loci were calculated, the number of alleles, effective number of alleles, Nei's genetic diversity and Shannon information index, and genetic relationship were analyzed by NTSYS pc version2.10e as well as the Nei’s genetic similarity coefficient (GS) and the genetic distance (GD). By using the characteristics, specific band of different primer combinations combined with Gel2.0 fingerprint automatic identification system, the apricot varieties (lines) in Xinjiang were identified, and the standard fingerprint database of apricot varieties (lines) was built using Gel2.0 fingerprint automatic identification system.【Result】Four ISSR primer pairs that with high polymorphism and good repeatability were selected from 100 ISSR primer pairs, and their names name respectively are UBC809, UBC836, UBC844 and UBC850 and they are regarded as specific primers. Four primer pairs produced 78 loci of which 73 were polymorphic. The percentage of polymorphic loci was 93.13%. UBC844 has the highest percentage of polymorphic loci of 100%. The average value of observed number of alleles, effective number of alleles, Nei’s genetic diversity and Shannon information index were 1.9313, 1.4368, 0.2622 and 0.4028, respectively, showing that the genetic diversity of all varieties (lines) was relatively abundant. The similarity coefficient of P. armeniaca cv. Huangrouyouxing and P. armeniaca cv. Dawuyuexing was 0.8846, genetic distance was 0.1226. The similarity coefficient of P. armeniaca cv. Kumanti and P. armeniaca cv. Suogejianali was 0.5641, and the genetic distance was 0.5725. Combine two different combinations of 4 primers could quickly and accurately identify 46 apricot varieties (lines). Only P. armeniaca cv. Lajiaoxing and P. armeniaca cv. Yiliakeyulvke need three primer combinations to be identified. A characteristic DNA fingerprint database of 48 apricot varieties (lines) was constructed by using the 4 ISSR primers with Gel2.0 fingerprint automatic identification system. 【Conclusion】The genetic diversity of apricot varieties (lines) in Xinjiang was relatively abundant and genetic relationship was relatively close. The genetic relationship of P. armeniaca cv. Huangrouyouxing and P. armeniaca cv. Dawuyuexing was the closest, but the genetic relationship of P. armeniaca cv. Kumanti and P. armeniaca cv. Suogejianali was the farthest. The results indicated that ISSR marker is suitable for the analysis of genetic diversity, relationship and construction of DNA fingerprinting database of Xinjiang apricot varieties (lines). Moreover, the fingerprinting database will provide a basis for identification, authenticity and purity of apricot varieties (lines), and provide a scientific basis for the registration, registration protection and intellectual property rights of new variety.

STORAGE·FRESH-KEEPING·PROCESSING

Effect of Germination-Extrusion-Amylase Assisted Processing on Quality Properties of Brown Rice Powder

ZHANG Dong-yuan, DENG Yuan-yuan, ZHANG Ming-wei, MA Yong-xuan, ZHANG Yan, WEI Zhen-cheng, ZHANG Rui-fen, LIU Lei, TANG Xiao-jun,TI Hui-hui

Scientia Agricultura Sinica. 2015, 48(4):  759.  doi:10.3864/j.issn.0578-1752.2015.04.13

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【Objective】The objective of this experiment is to investigate the effect of germination–extrusion–thermostable α-amylase assisted processing on quality properties of instant brown rice powder, such as solubility, fluidity, chromaticity, flavor and starch digestion performance and so on, thus providing the reference for processing of instant brown rice powder with high quality. 【Method】Germinated brown rice was extruded with thermostable α-amylase (EGBRE). Meanwhile, three control treatments were prepared: germination-exrusion processing (EGBR), thermostable α-amylase-extrusion processing (EBRE) and only extrusion processing (EBR). Characteristics of the four kinds of instant brown rice powder were investigated, including water solubility index, water absorption index, agglomeration rate, dispersion time, viscosity, chromaticity, volatile substances, performance of starch digestion and overall sensory evaluation. 【Result】 Thermostable α-amylase significantly improved the solubility of the brown rice powder. Compared with EGBR, water solubility index of EGBRE increased by 2.11 times, and water solubility index of EBRE increased by 2.55 times compared with EBR. However, germination didn’t affect the solubility significantly. Thermostable α-amylase could reduce the viscosity observably. Compared with EGBR, the viscosity of EGBRE decreased by 60.0%. The viscosity of EBRE decreased by 31.3% compared with EBR. Germination had no significant effect on the viscosity of the paste. Thermostable α-amylase increased the total content of volatile substances, but the content of lipid oxidation products in germinated samples was lower. The germination-extrusion-thermostable α-amylase processing could significantly reduce the agglomeration rate and shorten the dispersion time. Compared with EGBR, EBRE and EBR, the agglomeration rate of EGBRE was reduced by 55.4%, 74.8% and 84.0% respectively, and the dispersion time was shortened by 27.2%, 17.3% and 52.5%. Meanwhile, the co-processing could significantly influence the angle of repose, so its fluidity was improved. However, the chroma of the powder increased appreciably. Besides, germination-extrusion-thermostable α amylase processing improved the digestibility of starch. Compared with EBR, the proportion of fast-digesting starch of the EGBRE increased, and the ratio of resistant starch decreased significantly.【Conclusion】EGBRE could significantly improve the quality characteristics of instant brown rice powder. This co-processing increased the digestibility of starch and the content of volatile flavor compounds, meanwhile, decreased the rate of agglomeration, dispersion time and viscosity.

Effect on Immunogenicity of Pen a 1 and Its Epitopes Digested by Simulated Gastric Fluid

ZHAO Xin, GAO Mei-xu, MOU Hui, SHEN Yue, WANG Zhi-dong

Scientia Agricultura Sinica. 2015, 48(4):  769-777.  doi:10.3864/j.issn.0578-1752.2015.04.14

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【Objective】 Pen a 1 is a major allergen in shrimp, and its epitopes play a decisive role in allergic reactions. In order to evaluate the digestion stability of Pen a 1, the immunogenicity of Pen a 1 and its epitopes digested by simulated gastric fluid (SGF) was detected by antibodies of Pen a 1 and epitopes. It provided a theoretical basis for the study of the human digestive effects on Pen a 1 and the mechanism of desensitization food preparation.【Method】Pen a 1 was purified from Metapenaeus ensis. Five epitope peptides of Pen a 1 were synthesized using Fmoc Method. The peptides were conjugated to keyhole limpet (KLH) and bovine serum(BSA) to get artificial immunity and coating antigen, respectively. The tested serum was prepared by immuning New Zealand rabbits with Pen a 1 and the artificial immune. SGF was prepared according to the method of United States Pharmacopoeia. The changes of molecular weight of Pen a 1 was observed by SDS-PAGE and Tricine-SDS-PAGE. The capacity of IgE-binding of Pen a 1 and its epitope peptides by SGF digestion was analyzed by means of Western-blot qualitatively and ci-ELISA quantitatively.【Result】SDS-PAGE showed that Pen a 1 was degraded with increasing digestion time and generated a new 22 KD sensitization allergic protein. Tricine-SDS-PAGE showed that there were no protein fragments generated between 1.7-18 KD. Western-blot indicated that after the digestion of Pen a 1, the proteolytic fragments inordinately bound to six antibodies. Antibodies of Pen a 1 and No.4 bound to almost all proteolytic fragments. The fragment of 22 KD was resistant to digestion. This fragment bound to antibody No.3, 4, and 5 but almost no reaction to antibodies No.1, and 2, which indicated that the fragment carried No.3, 4, and 5 epitopes. The inhibition rate between Pen a 1 after SGF digestion and the antibodies tested by ci-ELISA was significantly decreased, indicating that the immunogenicity of Pen a 1 and its epitopes was decreased significantly. Moreover, the epitope peptides digested by SGF got the same result, but the digestion stability between epitopes of Pen a 1 and epitope peptides varied greatly: the epitopes of Pen a 1 was No.4＞No.2>No.1> No.3＞No.5, and there was no significant differences among No.3, 1, and 2; the epitope peptides was No.1> No.2> No.3＞No.4＞No.5, and there was no significant difference among No.2, 3, and 4. Combined with the results of Western-blot, No.4 epitope had the highest stability in SGF but the stability of No.4 peptide wasn’t higher than others significantly due to the protection of the spatial structure of Pen a 1. Stability of No.1, 2, and 3 was lower than No.4, and there was no significant differences between them. No.5 epitope showed rather rapid degradation in SGF than others.【Conclusion】The method for detection of digestibility of allergen and its epitopes using epitope antibodies was established. The immunogenicity of Pen a 1 after SGF digestion reduced significantly, but the generated fragments were still possible to cause hypersensitivity. No.4 epitope had the highest stability after SGF digestion, and No.5 epitope to SGF digestion was the most labile.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Analysis of Evolutionary Time of Porcine Endogenous Retrovirus in Guangxi Bama Mini-pigs

RAO Gui-bo, ZHONG Ya-ting, OUYANG Kang, MA Ling, HUANG Hong-mei, WU Jian-min

Scientia Agricultura Sinica. 2015, 48(4):  778-787.  doi:10.3864/j.issn.0578-1752.2015.04.15

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【Objective】The objective of this study is to determine the evolutionary time of the endogenous retrovirus (PERV-BM) in Bama mini-pigs.【Method】Analyses were performed on nine LTRs and one full-length genomic sequence of PERV-BM (accession number HM159246) amplified and sequenced by this lab and on the LTRs and env sequences of all PERV with clear backgrounds which have been published on GenBank. Firstly, through comparing the above sequences by using the DAMBE software, nucleic acid sequence data sets with high homology were combined and those that were typically different were screened. Then the phylogenetics of PERV-BM was calculated using the Neighbor-Joining method of MEGA5.2 software. DAMBE software was used to calculate the substitution saturations of the screened data sets and analyze the stability of their nucleic acid evolution rates. MEGA5.2 software was used to analyze the molecular clock of the phylogenetic tree built on the screened data according to their maximum similitude degree. Finally, substitution saturation and molecular clock analysis were done on the 3'-LTR sequences of the Bama miniature pigs in Guangxi closed group, then the sequences that were in consistence with the working of molecular clock were calculated by the hypothesized constant rate of evolutionary change in order to figure the evolutionary time of PERV-BM. 【Result】With all the analyses done with DAMBE software, the sequences of over 80 LTRs and env genes which are representatives in GenBank were selected. The phylogenetic analysis results of full-length sequence PERV-BM (HM159246) showed that it had closer genetic relationship with China PERV nucleic acid sequences EF133960 and GU980187 and Netherlands PERV nucleic acid sequences AF356697, but it was a bit distant from United States nucleic acid sequences AF038600 and NC_003059 and South Korea nucleic acid sequences HQ540595. Results of substitution saturation showed that the S and V values of the PERV sequences increased linearly with the increase of evolutionary divergence. The substitution saturation curve of LTRs sequence data sets had a linear relationship, without any substitution saturation; the evolution of LTRs was constant and stable with time. The substation saturation curve of env sequence data sets was not completely linear, indicating that the evolutionary rate of env sequence was unstable. The molecular clock test of the evolutionary tree established by the screened data sets with the maximum likelihood method by MEGA5.2 showed that LTRs sequence sets consisted with molecular clock hypothesis, while the evolutionary behavior of env did not meet the molecular clock. This was in accordance with saturation detection. With this regard, the approximate molecular clock behavior of LTRs could be used to calculate the evolutionary time of PERV-BM. Substitution saturation and molecular clock analysis on the 3'-LTR sequences of the Bama miniature pigs in Guangxi closed group showed that 3'-LTR sequence evolutionary behavior conformed to the molecular clock and could be used to calculate the evolutionary time. Supposed that the ERV mutation accumulation rate of the primate, the annual per nucleotide substitution rate 2.3×10^-9—5.0×10^-9, is used to calculate the evolutionary time, the result is that PERV-BM speciated approximately 3.3×10⁶—7.1×10⁶ years ago. 【Conclusion】The evolutionary time of PERV-BM is similar to that of the European mini-pigs which was 7.6×10⁶ years ago.

Effect of Chestnut Involucres Polyphenols on Growth Performance and Antioxidant Properties of AA Broilers

LI Hong, DONG Shuo, XIONG Ying, GU Ming-can, GUO Kai-jun

Scientia Agricultura Sinica. 2015, 48(4):  788-795.  doi:10.3864/j.issn.0578-1752.2015.04.16

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【Objective】Oxidative stress is widespread in livestock production. Lot of factors can induce oxidative stress, among which heat stress is an important factor. Oxidative stress can influence animal growth performance and even cause death, leading to serious economic losses. Recently, plant polyphenols are getting ever growing attention for their biological functions. The aims of this study were to investigate the effects of different doses of polyphenolic extract from involucres of Castanea mollissima Blume (PICB) on broiler growth performance and antioxidant properties. 【Method】 The experiments were divided into two trials: in trial 1, 400 one-day-old male Abor Aerec (AA) broilers were randomly assigned into 5 groups (4 replicates / group, 20 chicks / replicate): Group I was used as normal control group and the broilers were fed with basic ration; broilers in group II were fed with basic ration with a supplementation of 0.15% 2,6-Ditert butyl-4 methyl phenol (BHT) premix (containing 0.015% effective BHT); broilers in group III-V were fed with the basic ration with an addition of 0.2%,0.3% and 0.4% of PICB, respectively. Intrial 1, The trial lasted for 42 days under normal temperature condition. The broilers were reared according to the AA boilers feeding and immunity programs. The body weights and feed intake were measured every week for monitoring broilers growth performance. In trial 2, 400 28-day-old male AA chicks were randomly assigned into groups described in trial 1. After one week’s adaptation under normal temperature condition, heat stress was applied for 7 consecutive days for all groups. On 3rd and 7th day of heat stress, the chicks were slaughtered and sampled. Growth performance, slaughtering parameters and anti-oxidative parameters were measured to evaluate the effects of PICB on broilers under heat stress condition. 【Result】 Trial 1 showed that under normal temperature conditions, the supplementation of PCIB had no significant effects on chick growth performance (P＞0.05). In Trial 2, the body weights of groups Ⅳ and Ⅴ were significantly higher than that of group I under heat stress condition. Compared with groups I, II, and III, group IV had significantly improved average daily gain and average daily feed intake (P＜0.05). Feed conversion ratio of groups IV and V was significantly lower than that of group I (P＜0.05). The mortality among groups tended (P=0.059) to be significant and that of group V was the lowest. The slaughtering parameters had no significant differences among groups. For broilers heat stressed after three days, total antioxidant capacity (T-AOC) in serum was not significantly different among groups even though there were some oscillations. Total superoxide dismutase (T-SOD) of group IV was significantly higher than that of group I (P＜0.05). Serum malondialdehyde (MDA) contents of different groups were significantly different. The similar situation was also for GSH-Px except for group III significantly higher than group V (P＜0.05). 【Conclusion】 Dietary PICB supplementation had no effect on growth performance of AA broilers under normal temperature condition. While under heat stress conditions 0.3% PCIB addition can significantly improve the antioxidant capacity and growth performance of broilers and mitigate the adverse effects of heat stress.

Expression and Localization Analysis of the Odorant Receptor Gene Orco in Drones Antennae of Apis cerana cerana

ZHAO Hui-ting, GAO Peng-fei, ZHANG Gui-xian, TIAN Song-hao, YANG Shan-shan, MENG Jiao, JIANG Yu-suo

Scientia Agricultura Sinica. 2015, 48(4):  796-803.  doi:10.3864/j.issn.0578-1752.2015.04.17

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【Objective】 Insect odorant receptors (Ors) adopt a ligand-gated ion channel signal transduction mechanism primarily in olfaction recognition process. This ion channel is formed by a unique Orco protein with a common Ors which are combined to a heterodimer. Odorant receptors coreceptor (Orco) is an unique family, which shares highly conserved gene sequence among different insects and plays a key role in regulating insect behavior. The objective of this study is to research the expression character of Orco in drones of Apis cerana cerana based on the former relevant study in worker bees. 【Method】 Five different tissues of antennae, heads (without antennae), thoraces, abdomens and legs from 1-day-old drones, and the larvae, pupae and antennae of adults at different developmental stages were collected. Total RNA of each sample was extracted, and the expression profiles of the Orco in different tissues and at different developmental stages were examined by real-time quantitative PCR (qPCR). Antennae from 1-day-old drones were dissected and the frozen slides were prepared and the digoxigenin-labelled oligonucleotide probes were synthesized. The cellular localization in antennae was detected by in situ hybridization. 【Result】 qPCR results showed that the expression of Orco mRNA in different tissues were antenna>head>leg>abdomen>thorax, and it was about 100 times more abundant in antenna than in head. Orco transcripts were detected at all development stages of drones. The expression levels were very low in larval and pupal periods, increased after eclosion, then maintained a higher level during and after sexual maturity stage of drones. Combining with the former data got from the qPCR of worker bees, it showed that the expression level of Orco was significantly higher in drones than in workers. Localization test showed that Orco were largely expressed in neuronal cells of sensilla trihodea and sensilla placodea in antennae. 【Conclusion】 This study displayed the odorant receptor gene Orco was predominantly expressed in antennae of drones and the expression reached its peak when drones grow to sexual maturity. In addition, it was speculated combining the results of previous research that Orco expression biased in male sexually of A. cerana cerana.

RESEARCH NOTES

Structure Analysis of SSMP and Its Function in Salt Tolerance in Arabidopsis thaliana

ZHANG Hai-li, YOU Shi-dong, ZHANG Hao, GAO Jing, LI Sheng-hui, ZHANG Li-hui, XING Ji-hong, WANG Feng-ru, DONG Jin-gao

Scientia Agricultura Sinica. 2015, 48(4):  804-812.  doi:10.3864/j.issn.0578-1752.2015.04.18

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【Objective】The objective of this study is to analyze the structure of SSMP (salt-sensitive membrane protein), understand the expressing characteristics of SSMP, predict and analyze the function of SSMP in the process of stress resistance in Arabidopsis thaliana. 【Method】 The structure of SSMP protein was analyzed using bioinforatics method. Real-time PCR was adopted to analyze the temporal and spatial expression of SSMP in Arabidopsis. Preliminary analysis of the subcellular localization of SSMP was performed through the establishment of SSMP-GFP fusion expression vector and protoplast transformation. The tissue localization carrier was established with SSMP promoter connecting GUS reporter gene to analyze the tissue localization of SSMP. To determine the function of SSMP protein, overexpression experiments were performed in Col-0 Arabidopsis. Full-length cDNA was cloned into a binary vector downstream of the SSMP 35S promoter, and the construct was transformed into Col-0 Arabidopsis. Three lines positive seedlings of the transgenic Arabidopsis of over-expression of SSMP were obtained using Real-time PCR analysis. The phenotype and the stress tolerance of the transgenic Arabidopsis over-expression of SSMP were analyzed and so the biological function of SSMP gene could be understand. The cell membrane permeability was compared between wild type and the transgenic Arabidopsis over-expression of SSMP, the data of the cell membrane permeability was detected by the conductance instrument. The function of SSMP in the stress resistance process of Arabidopsis was analyzed.【Result】SSMP is a funtion unknown protein, it has 411 amino acids, containing a START conserved domain(the lipid/sterol-binding StAR-related lipid transfer protein domains). Bioinformatics analysis showed that SSMP has a phosphatidylcholine binding site, START domain could combine with lipids and sterols and transports cholesterol to inner membrane, so SSMP may influence the membrane components and play a role in the process of plant resistance. In this study, the expression level of SSMP in the tissues or organs of Arabidopsis was analyzed by using real-time PCR technique, andthe results showed that the order of the expression level of SSMP in Arabidopsis organs was stem leaf, cauline leaf, stem, root, flower and seed. Under normal circumstances, GUS is mainly expressed in hypocotyls, the cotyledon and true leaf veins and epidermal hairs. After treatment with 50 mmol·L^-1 NaCl, the expression of GUS in hypocotyls and cotyledons showed no significant changes, but the expression of GUS in true leaf were decreased. After treatment with 100 mmol·L^-1 NaCl, the expression of GUS was significantly reduced in the hypocotyl, cotyledon and true leaf, indicating that the expression of SSMP inhibited by NaCl. Preliminary analysis of the subcellular localization of SSMP was performed through the establishment of SSMP-GFP fusion expression vector and protoplast transformation and observed the fluorescence under 488nm wavelength using laser scanning confocal microscope. The result showed that SSMP was mainly localized on cytoplasma membrane. The cell membrane permeability of the transgenic Arabidopsis leaves of over-expression of SSMP was higher than that of the wild type. The expression of SSMP was inhibited and the germination of the seeds of SSMP function-gaining transgenic plants was lower obviously than wild type in the medium containing NaCl. 【Conclusion】SSMP is a protein of 411 amino acids with phosphatidylcholine binding sites, containing a START domain, mainly located on the cytoplasmic membrane, expressed in various tissues of Arabidopsis thaliana plants, especially the highest expression level in leaves. The expression of SSMP mainly localized in the genesis areas of leaf vein and epidermal hair. The expression of SSMP was inhibited by salt stress. The cell membrane permeability increased and the salt tolerance decreased in Arabidopsis overexpressing SSMP.

Effects of Post-Anthesis Waterlogging, High Temperature and Their Combination on Starch Compositions and Pasting Properties in Wheat Grains

WANG Chen-yang, ZHANG Yan-fei, LU Hong-fang, ZHAO Jun-xia, MA Geng, MA Dong-yun, ZHU Yun-ji, GUO Tian-cai, MA Ying, JIANG Yu-mei

Scientia Agricultura Sinica. 2015, 48(4):  813-820.  doi:10.3864/j.issn.0578-1752.2015.04.19

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【Objective】 Waterlogging(WL) and high temperature(HT) are major meteorological disasters to winter wheat after anthesis in both the middle and lower Yangtze Valley and the south of Huanghuai wheat production regions, which have a great impact on grain yield and quality of winter wheat. However, little is known on the effect of various stresses and their combination(WL+HT) on starch formation and quality in wheat grains. 【Method】 A pot experiment was conducted in the growing season of 2011-2013. Wheat cultivar Zhengmai 004, was treated with waterlogging and/or high temperature stress from the 5th to 14th day after anthesis. 【Result】The starch yield was reduced significantly under WL, HT and WL+HT stresses. The reduction percentages were 28%, 46% and 52%, respectively in 2011-2012 year and 27%, 43% and 61% in 2012-2013 year, respectively. Although slight increase of amylopectin concentration and decreases of amylose concentration and the ratio of amylose to amylopectin were observed in the experiment, there was no significant difference existed in starch compositions between WL and CK group. HT and WL+HT decreased the concentrations of amylose and amylopectin, and significantly decreased the concentration of total starch by 11.0% and 10.8%, respectively in 2011-2012 growing season, and by 8.2% and 5.5%, respectively in 2012-2013 growing season. WL significantly decreased the minimum viscosity(MV) in 2011-2012 growing season, but had no significant impact on any other pasting parameters. However in the growing season of 2012-2013, WL significantly increased the peak viscosity (PV) minimum viscosity(MV) , final viscosity(FV), breakdown and setback by 27.9%, 39.2%, 31.5%, 21.9% and 27.9%, respectively. HT significantly reduced the peak viscosity(PV) , minimum viscosity(MV) , final viscosity (FV) , breakdown and peak time(PT) by 22.2%, 22.0%, 16.9%, 22.5% and 4.8%, respectively in 2011-2012 growing season, and by 39.6%, 62.0%, 62.7%, 28.0% and 7.0%, respectively in 2012-2013 growing season. Effects of WL+HT combination on starch pasting properties differed between the experimental seasons. WL+HT significantly decreased PV, MV, FV and breakdown by 20.9%, 23.9%, 10.4% and 15.2%, respectively in 2011-2012 growing season, but increased by 15.4%, 30.2%, 7.1% and 6.4%, respectively in 2012-2013 growing season. Correlation analysis indicated that both the content of the total starch and amylopectin were positively correlated with PV, MV, FV and breakdown in both wheat cultivars. However, no significant correlations were obtained between the content of amylose and pasting properties in the experiment. 【Conclusion】 Post-anthesis WL and HT significantly reduced starch yield. And the combination stress of WL and HT(WL+HT) increased the damage. WL, HT and WL+HT strongly influenced starch content and its compositions, and resulted in obvious changes of starch pasting properties. In the current study, we also found that HT had a relatively greater influence on starch quality than that of WL. However, the combination stress didn’t improve the impact of HT on pasting properties, indicating that interaction existed between WL and HT.

The Development of Frizzled Follicle and Genetic Characteristics of Candidate Gene KRT75 in Frizzled Feather Chicken

TAO Lin, DU Bing-wang, ZHANG Li

Scientia Agricultura Sinica. 2015, 48(4):  821-830.  doi:10.3864/j.issn.0578-1752.2015.04.20

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【Objective】The Objective of this study is to analyzes the features and curve process of frizzled feather, the development of frizzled feather follice, sequence features and expression rules of candidate gene KRT75 in frizzled feather follicle development. 【Method】 The frizzled feathers of 360 frizzled chicks from 1 to 300 days were observedto know the curve process, and the microstructure of rachis, barbules, anf hooks between frizzled and contour feather were compared. The development of feather follicle between frizzled feather and contour feather at 8th in embryonic period was compared by Microscopic section technology. The total RNA from the embryonic 13 day skin of frizzled feather and contour feather was extracted, respectively, reverse transcripted cDNA，connected with the PMD-18T vector，the products were transformed into DH5α competent cells, coated plates, monoclonal colony was selected for PCR, and the positive plasmid was chosen for sequencing. In addition, 15 frizzled and contour feather chicks at 300 days were randomly selected to collect vein blood under the wing, blood genome pillar extraction kit was used to extract DNA, the PCR products were also sequenced after purification, and the DNAMAN software was used to analyze genetic characteristics of KRT75 gene. The total RNA was also extracted from embryonic 8 to 17 day skin of frizzled feather and contour feather; respectively, reverse transcripted cDNA, and the SYBR kit was used to analyze KRT75 gene expression during the formation of feather follicle. 【Result】 The down feather appeared disorder and curved in newborn frizzle chicks, the largest curvature of feather was observed at 6 weeks. The frizzled feather rachis curved outward, barbules could not hooked together to form a closed feather; the development of frizzled feather follicle and contour feather follicle was both from E8 to E16, there were some differences in the medulla and barb ridge between frizzled feather and normal one from E12 to E15, in the E12 to E14 day, the medulla of frizzled feather were bigger than contour feather, but the barbule plates were smaller than contour feather. The CDS of KRT75 gene was 1 569 bp both in frizzled feather and contour feather chickens, no 69 bp deletion mutation was detected in CDS region of frizzled chicken KRT75 gene. Three SNPs ( 954bp: T>C; 967bp: T>C; 978bp: C>T) were found compared frizzled chicken with Princess chicken. The expression of KRT75 was significantly different between frizzled feather and contour feather, KRT75 was low expression in contour feather follice but was highly expressed in frizzled feather follicle from E9 to E17, especially in E9,E10 and E12 to E15, the KRT75 of frizzled feather was highly significant than contour feather(P<0.01). 【Conclusion】 Frizzled feather of KiRin Chicken was not caused by 69 bp detection mutation of KRT75 gene. The three SNPs (955bp: T>C; 967bp: T>C; 978bp: C>T) could be used as molecular markers in distinguishing frizzled and contour feathers.