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    16 April 2019, Volume 52 Issue 8
    MALE STERILITY OF CROP
    The Function of the Polyketide Synthase OsPKS1 and OsPKS2 in Regulating Pollen Wall Formation in Rice
    ZHOU YuLu,LIN Hong,ZHANG DaBing,WANG CanHua,YU Jing
    Scientia Agricultura Sinica. 2019, 52(8):  1295-1307.  doi:10.3864/j.issn.0578-1752.2019.08.001
    Abstract ( 665 )   HTML ( 94 )   PDF (6571KB) ( 408 )   Save
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    【Background】 Plant pollen is surrounded by pollen wall which acts as a natural protective barrier for male gametes and plays a pivotal role in plant reproductive development. The main component of pollen wall is sporopollenin, which is mainly composed of lipidic and phenolic substances. Therefore, the metabolism of these two substances is a key step for anther wall and pollen wall formation. PKS1/PKSA/LAP6 and PKS2/PKSB/LAP5 show conserved biochemical functions in sporopollenin biosynthesis pathways among different species. 【Objective】 The role of OsPKS1 and OsPKS2 in rice anther wall and pollen wall development was studied to provide a new understanding for the mechanism of this process. 【Method】 A gene co-expressed network, AntherNet predicted a gene OsPKS1 that might be involved in sporopollenin biosynthesis, using the CRISPR/Cas9 genome editing system to generate ospks1 and ospks1 ospks2 in Japonica subspecies 9522 background and ospks2 background, respectively. Under the same growth condition, the vegetative growth and floral organ development of the mutant plants were analyzed by comparing the phenotypes of the wild type and the mutants. I2-KI staining was utilized to analyze the pollen viability of ospks1 and ospks1 ospks2. Semi-thin section was performed to observe four cell layers and microspore development in the wild type and the mutants at different stages. Scanning electron microscope (SEM) was used to observe the fine structures of anther wall outer and inner surface as well as pollen wall surface both in the wild type and the mutants. And transmission electron microscopy (TEM) was performed to observe the fine structures of anther wall cell, Ubisch body and pollen wall of the wild type and the mutants.【Result】Four ospks1 and four ospks1 ospks2 were obtained by CRISPR/Cas9 approach, among which ospks1-3 and ospks1-4 ospks2 were homozygous mutants. Both ospks1-3 and ospks1-4 ospks2 were male sterile. ospks1-3 and ospks2 displayed abnormal pollen wall and Ubisch body, however, the detailed morphology was different between two mutants. ospks1-3 could form convex wall structure on the surface of pollen wall and the tapetal layer could be normally degraded; a large number of tiny cavities were formed in the inner structure of pollen wall and the bacula became shorter, which might cause invalid connection between tectum and nexine; the bottom structure of Ubisch bodies was decreased while the top structure was increased and Ubisch bodies were sharper than those of the wild type. By observing ospks1-4 ospks2, it was found that the cuticle was reduced and the tapetal layer could not be degraded normally. Besides, there was no obvious pollen wall structure on the surface of microspores and the microspores were degraded at stage 11; Ubisch bodies were less formed with abnormal structure at stage 9 and were detached from the anther wall at stage 11. 【Conclusion】 The function of PKS1/PKSA/LAP6 and PKS2/PKSB/LAP5 are conserved among various species and could affect the biosynthesis and accumulation of sporopollenin. However, these two genes show different function for the formation of the inner structure of pollen wall and Ubisch bodies in rice: OsPKS1 is more important for the formation of bacula and the bottom structure of Ubisch bodies; OsPKS2 is more important for the formation of tectum and the top structure of Ubisch bodies. They regulate the formation of pollen wall, anther wall and the degradation of tapetum.

    Comparative Transcriptome Analysis Among the Three Line of Cytoplasmic Male Sterility in Maize
    XUE YaDong,YANG Lu,YANG HuiLi,LI Bing,LIN YaNan,ZHANG HuaiSheng,GUO ZhanYong,TANG JiHua
    Scientia Agricultura Sinica. 2019, 52(8):  1308-1323.  doi:10.3864/j.issn.0578-1752.2019.08.002
    Abstract ( 645 )   HTML ( 49 )   PDF (2081KB) ( 488 )   Save
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    【Objective】It is one of the most efficient ways to utilize cytoplasmic male sterile (CMS) lines in hybrid seed production, which could improve the purity of seeds, reduce the cost in creating hybrid seeds and enhance the competitiveness of Chinese seed companies. The comparative transcriptome analysis of the anthers at different development stages from the CMS line, the maintainer line and the restorer line (the three lines) were performed in order to understand the mechanism of sterility and restoration of CMS-C in maize, and also to elucidate the regulation network between the restorer gene and the sterile gene, which will provide the fundamental basis for the employment of maize CMS in hybrid seed production.【Method】The transcriptome sequencing was carried out on the anthers at the prophaseⅠ, the metaphaseⅠand the tetrad stage from the three lines based on the elite inbred line Yu87-1. Method of comparative analysis was used to deal with all the transcripts by the tools such as hisat2, ballgown and DESeq2, and to predict genes involved in the regulation network between the sterile gene and the restorer gene, between the different development stages and through the development time series. qRT-PCR was used to verify the differentially expressed genes. The activity of ATPase was quantified with by the spectrophotometric method for the verification of the putative hypothesis.【Result】Transcriptome sequencing totally produced 156.59 Gb sequence data. After mapping and assembling, 53035 Unigenes were obtained. A total of 5676 differentially expressed (DE) genes were identified from the pairwise comparisons (except for comparisons between the restorer lines and the maintainer lines) in the anthers at the different stages from the three lines. Of those, 4705 DE genes between the comparisons of the development stages, 2693 DE genes between the comparisons of the different lines and 135 DE genes related to the time series. The GO molecular functional analysis showed that the genes related to ATP binding, DNA binding and zinc ion binding were highly enriched, in cell component analysis, genes located in integral component of membrane, nucleus and plasma membrane were enriched, and in biological process, genes involved in DNA-templated transcription, regulation of transcription, oxidation-reduction process and primary metabolic process were enriched. KEGG pathway analysis indicated that the oxidative phosphorylation pathways, the carbon metabolism pathways and glycolysis pathways were mostly enriched. Compared to the maintainer lines, several genes involving in the oxidative phosphorylation pathways were significantly down-regulated in the sterile lines, while those down-regulated genes were recovered, besides other genes in the same pathways were also coordinately regulated. The expression trend determined by qRT-PCR on the selected DE genes was in accordance with that in the transcriptome data. The enzyme activity results show that the activity of ATPase in the sterile line was greatly reduced compared to the maintainer line, while in the restorer line, the activities the ATPase were restored due to the existence of the restorer gene.【Conclusion】 The onset of the changes in the gene expression caused by the sterile gene in the anthers of CMS-C maize may happen after metaphaseⅠ and before telophase Ⅱ in meiosis before visible phenotype occurred. The energy deficiency model may account for the mechanism of the sterility in maize CMS-C, and the energy requirements were compensated by the restorer gene through direct or indirect manner.

    Effects of Female Fertility of RN Type Male-Sterile Lines on Outcrossing Rate
    ZHANG JingYong,YAN Hao,PENG Bao,ZHANG ChunBao,LI Hui,WANG PengNian,DING XiaoYang,LIN ChunJing,SUN Huan,ZHAO LiMei,ZHANG Wei
    Scientia Agricultura Sinica. 2019, 52(8):  1324-1333.  doi:10.3864/j.issn.0578-1752.2019.08.003
    Abstract ( 365 )   HTML ( 36 )   PDF (378KB) ( 312 )   Save
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    【Objective】To study the differences in female fertility of RN-type soybean cytoplasmic male sterile lines (A-lines) and maintainer lines (B-lines), determine whether there is a decline in female fertility in RN-type soybean cytoplasmic male sterility lines, and explore the correlation between female fertility and outcrossing rate in male sterile lines. 【Method】 Firstly, 6 pairs of sterile lines and maintainer lines were selected from more than 200 RN-type cytoplasmic male sterile lines; outcrossing rates among these lines were different. Second, the outcrossing rate of male sterile lines was evaluated using honeybee pollination in net cages. Third, six male sterile lines were pollinated by non-removing stamen test, with male parents of the same restorer line, to determine whether there were differences in fertilization and podding. Finally, the differences in survival rate between sterile lines and maintainer lines were studied by removing stamens and non-removing stamens parallel cross test with the same restorer line used as the male parent. 【Result】 The outcrossing rates in cages were significantly different among the six sterile lines tested: the highest outcrossing rate was 49.46% and the lowest outcrossing rate was 15.94%. there were significant differences among survival rates of artificial crosses of six sterile lines tested: the survival rate of sterile lines with a high outcrossing rate were significantly higher than that of medium and low outcrossing rate sterile lines and the survival rate of sterile lines with medium outcrossing rate were significantly higher than that of low outcrossing rate sterile lines. For crossing survival rate after artificially removing stamens, A-lines with high and medium outcrossing rates were significantly higher than that with low outcrossing rates; there were no significant differences among B-lines with high, medium, or low outcrossing rates; there were no significant differences among high A-lines, middle A-lines, and their corresponding B-lines, while A-lines with a low outcrossing rate were significantly lower than their corresponding B-lines. For crossing survival rate by artificially not removing stamen, A-lines with a high outcrossing rate were significantly higher than that with medium or low outcrossing rates; there were no significant differences among B-lines with high, medium and low outcrossing rates; there were no significant differences among high A-lines, middle A-lines, and their corresponding B-lines, while A-lines with low outcrossing rate were significantly lower than their corresponding B-lines. 【Conclusion】 For RN-type cytoplasmic male-sterile lines of soybean, female fertility of A-lines with high outcrossing rates were normal and female fertility of a few A-lines with low outcrossing rates were poor, affecting their seed setting. Female fertility of B-lines with different outcrossing rates were all normal, the outcrossing rate of A-lines in cages were significantly positively correlated with their crossing survival rate following artificial stamen removal and in plants where the stamen was not removed, the outcrossing rates of A-lines in cages were not correlated with their B-lines’ crossing survival rate. All parallel crosses by removing and non-removing stamens can thus be used to evaluate female fertility of A-lines.

    A Simplified Production Method of Hybrid F1 Seeds in Rapeseed
    YANG GuangSheng,XIN Qiang,DONG FaMing,HONG DengFeng
    Scientia Agricultura Sinica. 2019, 52(8):  1334-1340.  doi:10.3864/j.issn.0578-1752.2019.08.004
    Abstract ( 404 )   HTML ( 21 )   PDF (309KB) ( 327 )   Save
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    Safe and effective F1 seed production method is the key to hybrid production in rapeseed. Father parent lines with tribenuron-methyl resistance (or tolerance) and self-incompatiblity can be developed by means of backcross breeding. In the F1 hybrid seed production by using tribenuron-methyl as the male sterility induction agent, the father parent lines have no seedset by self-pollination because of their self-incompatibility, and are not affected by the chemical agent because of their tribenuron-methyl resistance (or tolerance). Therefore, the parental lines can be mixed sowing and the seeds can be mixed harvesting. Using the method we proposed, if the lines with tribenuron-methyl resistance (tolerance) and self-incompatiblity are already developed, hybrid breeding can be carried out immediately. It saves labor costs, improves seed production efficiency and increases seed purity because of the mixed sowing and mixed harvesting.

    Overview of the Study and Application of Cytoplasmic Male Sterility in Cotton
    WANG XueDe
    Scientia Agricultura Sinica. 2019, 52(8):  1341-1354.  doi:10.3864/j.issn.0578-1752.2019.08.005
    Abstract ( 457 )   HTML ( 29 )   PDF (469KB) ( 374 )   Save
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    Cotton has significant heterosis. Hybrid cotton usually can increase production in lint yield by about 15% compared with conventional self-pollinated cultivars, and also can get obvious improvement in fiber quality, disease resistance, insect resistance, adversity resistance and photosynthetic efficiency. Among some links of cotton heterosis use, the most important link is the castration in the production of hybrid seeds. At present, there are four ways for the castration, such as hand emasculation, chemical male gametocide, nuclear male sterility and cytoplasmic male sterility (CMS). The production practice showed that use of cotton male sterility could not only simplify the hybrid seed production but also save the production cost on a commercial economic scale. In particular, the way of hybrid seed production by use of cotton CMS line, maintainer line and restorer line were the most effective way since it could overcome some disadvantages in the other ways. Therefore, in this paper, the study and application of the cotton CMS system in hybrid seed production were overviewed and some of problems currently limiting application were also addressed. At first, the genetic, cytological and biochemical characteristics of the cotton CMS were reviewed. Secondly, the positive/negative effects of sterile cytoplasm in hybrid F1 were analyzed, and how to overcome these negative effects, such as pollen temperature sensitive and F1 not expressing complete fertility, by developing strong restorer lines with a stronger ability for F1 fertility restoration, was discussed in detail. For an example, transgenic strong restorer line could be developed by introducing the exogenous GST gene, which was assumed to have the function of enhancing pollen vitality, into some conventional restorer lines, and so that hybrids with higher heterosis could be produced by crossing this strong restorer with sterile lines. According to the characteristics of cotton as an often cross-pollination crop, this paper recommended in detail the key techniques of hybrid cotton seed production, such as rules of parent (sterile line and restorer line) selection, location selection and environment optimization for enriching native pollinators to produce more hybrid seeds. Then, the paper pointed out that compared with other crops, cotton hybrid seed production based on CMS system has four advantages in the cotton heterosis use: (1) The purity of hybrid seeds can be guaranteed because there is no pollen in anthers of cotton CMS line and its sterility is very stable and not affected by the climate and other environments; (2) The high yield of hybrid seed can be obtained since the long flowering period (about 3 months) of cotton does not result in the flowering asynchronism between sterile line and restorer line; (3) The wide ecological adaptability of cotton and the possibility of large-scale hybrid seed production will be benefited to popularize hybrid cotton; and (4) Interspecific heterosis between upland cotton (Gossypium hirsutum L.) and sea-island cotton (G. barbadense L.) can be used. It is predicted that the hybrid cotton production based on CMS system will be the main approach to utilize heterosis of cotton. Finally, the future works in study and application of CMS in cotton heterosis, especially in development of new sterile lines and restorer lines by use of modern biotechnology, was also discussed.

    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Analysis of Gap Between Yield and Radiation Production Efficiency and Temperature Production Efficiency in Summer Maize: Taking Shandong Province as an Example
    WANG HongZhang,LIU Peng,DONG ShuTing,ZHANG JiWang,ZHAO Bin,REN BaiZhao
    Scientia Agricultura Sinica. 2019, 52(8):  1355-1367.  doi:10.3864/j.issn.0578-1752.2019.08.006
    Abstract ( 449 )   HTML ( 38 )   PDF (611KB) ( 465 )   Save
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    【Objective】 In the present study, the biomass production and resource availability among yield levels were studied to quantify the gap of yield, radiation production efficiency and temperature production efficiency of summer maize in Shandong province. This study aimed to clarify the contribution rate of agricultural production conditions and cultivation measures to yield gap and efficiency gap, and to explore the possibility of synergistic narrow the yield gap and efficiency gap, so as to provide a theoretical basis for closing yield gap and improving resource utilization efficiency. 【Method】 The experiment was conducted in Taian, Zibo and Yantai in Shandong province from 2017 to 2018. Based on the investigation of summer maize production in Shandong province, four management models were designed in consideration of appropriate increase of plant density, optimization of fertilizer and water, increase of yield and efficiency with the same integrated management. The four yield levels, including super high yield (SH), high yield and high efficiency (HH), farmer level (FP) and basic production level (CK), were simulated. And the gap of yield, radiation production efficiency and temperature production efficiency of different yield levels were analyzed. With the integrative analysis of radiation-temperature production potential and crop yield performance, the factors affecting gap of yield and efficiency and the way closing yield gap and increasing efficiency were explored in the present study. 【Result】 At present, the yield gap between radiation temperature potential level and super high yield level, super high yield level and high yield high efficiency level, high yield and high efficiency level and farmer production level, farmer production level and basic production level of summer maize in Shandong province were 5.85, 0.82, 1.90 and 1.35 t·hm -2, respectively; The radiation production efficiency gap were 1.74, 0.15, 0.28 and 0.45 g·MJ -1, respectively; and the temperature production efficiency gap were 1.09, 0.10, 0.17 and 0.28 kg·hm -2·℃ -1, respectively. The current uncontrollable factors contributed 58.49% to yield gap, and contributed 66.09% to light and temperature production efficiency. And geographical difference factors contributed 1.98% to yield gap, contributed 2.49% to radiation production efficiency, and contributed 3.24% to temperature production efficiency. There was a significant correlation between the yield gap and the production efficiency gap. SH and HH had higher biomass, mean leaf area index (MLAI) and canopy light energy interception rate than FP and CK. 【Conclusion】 At present, the gap of yield, the radiation production efficiency, and the temperature production efficiency between the farmer production level and the radiation temperature potential level of summer maize in Shandong province were 8.56 t·hm -2, 2.17 g·MJ -1, and 1.35 kg·hm -2·℃ -1, respectively, so there was room for improvement in yield and utilization efficiency of radiation and temperature resources. There was a significant correlation between the yield gap and the production efficiency gap, on the basis of existing farmer management measures, the application of high-yield and high-efficiency management mode (increase the plant density of 15 000 plant·hm -2, and increasing the amount of fertilization appropriately, changing the one-time fertilization into the sub-fertilization mode with water and fertilizer integration during the stage of sowing, spike formation, flowering, and milking) could narrow the yield gap by 1.90 t·hm -2and increase the production efficiency of radiation and temperature resources by 14.74% and 14.41%, respectively, which was an effective technical way to synergistic close yield gap and increase efficiency.

    Effects of Fertilization Rate on Forage Yield and Water Use Efficiency of Artificial Grassland in an Alpine Arid Area
    ZHANG XueMei,MA QianHu,ZHANG ZiLong,WANG ZiKui,YANG HuiMin,SHEN YuYing
    Scientia Agricultura Sinica. 2019, 52(8):  1368-1379.  doi:10.3864/j.issn.0578-1752.2019.08.007
    Abstract ( 444 )   HTML ( 21 )   PDF (524KB) ( 282 )   Save
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    【Objective】 This study was conducted to investigate the production of artificial grassland and the response of water resource use to planting pattern and fertilization level, so as to provide the optimal planting pattern and fertilization level in the desert steppe region of the Qinghai-Tibet plateau. 【Method】 The field experiment was conducted at Jintai pasture of Wulan county, Qinghai province in 2017 and 2018. Two planting patterns were set up, namely oat/common vetch mixed sowing and sole oat, and two fertilization rates were set up, which were high rate (120 kg N·hm -2, 103 kg P2O5·hm -2) and low rate (60 kg N·hm -2, 51.5 kg P2O5·hm -2). Four treatments were completely randomized block designed. All treatments were irrigated before sowing in the spring and no irrigation during the growing season. The measured indexes included aboveground biomass, plant nitrogen content and soil water content at major growth stage; The calculated indexes included water consumption during the growing stage, dry matter water use efficiency, and crude protein water use efficiency. 【Result】 Through mixed sowing and adding fertilizer, grassland yield could be greatly improved. In 2017 and 2018, the dry matter yield of high-fertilizer treatment was 34.7% and 9.7% greater than that of low-fertilizer treatment, respectively, and the crude protein production was 41.3% and 20.4% greater, respectively. The dry matter yield of oat/common vetch mixed sowing was 14.4% and 9.2% greater than of sole oat treatment, respectively, and the crude protein yield was 74.7% and 62.9% greater, respectively. The yield of mixed sowing with high-fertilizer treatment was the highest under all treatments, the dry matter and crude protein yield at harvest were 10 251.7 and 827.2 kg·hm -2, respectively, in 2017, and 7 589.3 and 570.2 kg·hm -2, respectively, in 2018. Adding fertilizer increased the water consumption of grassland, and the water use efficiency of grassland was greatly improved. In the two growing seasons in 2017 and 2018, the water consumption of high-fertilizer treatment was 6.2% and 4.3% higher than that of low-fertilizer treatments, respectively, the water use efficiency of dry matter increased by 21.0% and 4.9%, respectively, and the water use efficiency of crude protein was increased by 30.1% and 17.4%, respectively. Mixed sowing also increased the water consumption of grassland, slightly reduced the water use efficiency of dry matter, but greatly improved the water use efficiency of crude protein. In 2017 and 2018, the water consumption of oat/common vetch mixed sowing grassland was 17.9% and 9.2% higher than that of sole oat grassland, respectively; The water use efficiency of crude protein was increased by 46.7% and 51.3%, respectively.【Conclusion】Mix sowing and rational fertilizer application could increase the forage yield and quality as well as water use efficiency of grassland by decreasing the ineffective evaporation, and enhance the utilization of soil water. Which was recommended to be applied with spring irrigation conditions in the arid alpine area, the research can provide some reference for the cultivated grassland and excellent forage production in alpine arid and semi-arid areas in China.

    PLANT PROTECTION
    Construction and Evaluation of ATMT Mutant Library of Fusarium verticillioides
    SUN Hua,MA HongXia,DING MengJun,LI Po,SHI Jie,LIU ShuSen
    Scientia Agricultura Sinica. 2019, 52(8):  1380-1388.  doi:10.3864/j.issn.0578-1752.2019.08.008
    Abstract ( 395 )   HTML ( 13 )   PDF (1488KB) ( 178 )   Save
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    【Objective】The objective of this study is to establish a highly efficient ATMT mutagenesis system of Fusarium verticillioides, and to construct ATMT mutant library in which mutant contains green fluorescent protein (GFP). And then this library was used for screening and analysis, which can lay a foundation for studying the infection pathway and molecular pathogenesis of F. verticillioides on maize ear. 【Method】 The inhibitory concentration of cefotaxime sodium (Cefo) and ampicillin sodium (Amp) against Agrobacterium tumefaciens AGL-1 and the sensitive concentration of hygromycin B against F. verticillioides were screened for ATMT mutagenesis system. A shuttle plasmid containing GFP and hygromycin phosphotransferase (HPH) genes was used as a vector to construct the ATMT mutants library of F. verticillioides. The T-DNA insertion and stability of transformant were detected and analyzed through hygromycin B resistance, PCR identification of GFP-specific primers, and fluorescence microscopy. Nine transformants were randomly selected and the sporulation number, conidial germination rate, and pathogenicity were measured. 【Result】When the concentration of Cefo/Amp was 150/150 μg·mL -1, the growth of AGL-1 was inhibited, and when the concentration of hygromycin B was 150 μg·mL -1, the growth of F. verticillioides was completely incapacitated. Using the optimized ATMT, a total of 2 465 GFP-labeled transformants were obtained, these transformants could still grow normally on PDA medium containing hygromycin B after cultured 5 generations on hygromycin-free PDA medium, which indicated that HPH had been integrated into wild-type (WT) genome of F. verticillioides and the transformants were stable in their characteristics of genetics. The PCR detection results amplified with GFP-specific primers showed that the homology of transformants with GFP (accession number: LC420351.1) in NCBI was 99.26%, and the hyphae and conidia of transformants showed green fluorescence under the fluorescence microscope, while no fluorescence was observed in WT, indicating that GFP had been integrated into the WT genome and successfully expressed. Compared with WT strain, the sporulation number of transformant 54 increased significantly, about 1.9 times of that of WT strain, and the conidial germination rate of transformant 24 decreased obviously in the same time. The pathogenicity of transformant 13 was enhanced, the disease grade reached 9, the pathogenicity of transformant 33 and 16 was reduced to grade 3, and the pathogenicity of transformant 4 was the weakest, the disease grade was 1. There was no significant change in biological characters of partial transformants.【Conclusion】ATMT mutant library with GFP of F. verticillioides was constructed, and the mutants with changed sporulation number, conidia germination rate and pathogenicity were obtained through primarily screening. It will lay a foundation for further study on the infection pathway and pathogenic molecular mechanisms of F. verticillioides on maize ear in the future.

    Enzymatic Characteristics and Metabolic Analysis to Malathion and p,p’-DDT of LmGSTS2 from Locusta migratoria
    MA Wen,LIU Jiao,ZHANG XueYao,SHEN GuoHua,QIN XueMei,ZHANG JianQin
    Scientia Agricultura Sinica. 2019, 52(8):  1389-1399.  doi:10.3864/j.issn.0578-1752.2019.08.009
    Abstract ( 293 )   HTML ( 14 )   PDF (1008KB) ( 173 )   Save
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    【Objective】 Glutathione S-transferase sigma2 (LmGSTS2) from Locusta migratoria was expressed in Escherichia coli and purified in order to analyze the enzymatic characteristics. The objective of this research was to study the effect of LmGSTS2 on malathion and p,p’-DDT metabolism. The detoxification ability of LmGSTS2 was assessed by using LmGSTs2 RNA interference (RNAi) and insecticide bioassay. It will provide a theoretical basis for management of locust resistance and rational insecticide application.【Method】LmGSTS2 was expressed in BL21 (DE3) cells and purified by Ni-NTA affinity chromatography. The activities of LmGSTS2 under different conditions (temperature and pH) were detected using CDNB as substrate. Under the optimal conditions (pH 7, 27℃), malathion and p,p’-DDT were incubated with purified LmGSTS2 protein. The metabolic detoxification ability of LmGSTS2 to malathion and p,p’-DDT was evaluated by ultra performance liquid chromatography (UPLC). Furthermore, 3 μg of dsLmGSTs2 was injected into 2nd instar nymph, RNAi efficiency of LmGSTs2 was tested at 24 h after dsLmGSTs2 injection and the sensitivity of L. migratoria to malathion was analyzed at 24 h after malathion exposure. 【Result】 LmGSTs2 was induced to express in E. coli. After SDS-PAGE detection, it was found that an extra band around 25 kD in the total protein of pET28a/BL21 (DE3)-LmGSTS2 after induction compared with pET28a/BL21(DE3) and uninduced pET28a/BL21(DE3)-LmGSTS2, which is regarded as the target protein size, indicating that LmGSTS2 was successfully expressed in the bacteria. The results of the study on the enzymatic characteristics of the purified LmGSTS2 protein showed that the optimum reaction pH was 6-8, the enzyme activity reached the peak at pH=7, the optimum reaction temperature was 25-30℃, and the activity was the highest at 27℃. LmGSTS2 was exposed to malathion and p,p’-DDT respectively at pH 7, 27℃. The results of UPLC showed that the peak area of malathion after incubation with LmGSTS2 decreased by 83.6%, 84.0% and 84.6%, respectively, compared with GSH+insecticide, active LmGSTS2+insecticide and inactive LmGSTS2+GSH+insecticide (P<0.05). However, there was no significant change in the peak area of p,p’-DDT compared with the control group (P>0.05), indicating that LmGSTS2 could metabolize malathion, but had no effect on the metabolism of p,p’-DDT. The role of LmGSTS2 in the detoxification process of malathion was further verified by RNA interference. The dsRNA of the target gene was injected into the 2nd instar nymph. After 24 h, the mRNA expression of LmGSTs2 was inhibited by 96%. The sensitivity test showed that compared with the control group, the sensitivity of L. migratoria to malathion increased after gene silencing, and the mortality increased from 29.9% to 45.2%, indicating that LmGSTS2 was involved in the detoxification process of malathion in L. migratoria. 【Conclusion】 LmGSTS2 was expressed and purified in vitro, and the optimal reaction condition of the enzyme was pH=7, 27℃ using CDNB as substrate. In vivo and in vitro assays for malathion metabolism showed that LmGSTS2 was involved in the metabolic detoxification in L. migratoria. In vitro assay for p,p’-DDT metabolism showed that LmGSTS2 was not involved in the metabolism of p,p’-DDT.

    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Quantitative Analysis of Driving-Factors of Soil Acidification in Qiyang County, Hunan Province
    ZHOU HaiYan,XU MingGang,CAI ZeJiang,WEN ShiLin,WU HongHui
    Scientia Agricultura Sinica. 2019, 52(8):  1400-1412.  doi:10.3864/j.issn.0578-1752.2019.08.010
    Abstract ( 480 )   HTML ( 11 )   PDF (530KB) ( 347 )   Save
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    【Objective】In Qiyang County, Hunan Province which is a typical county of China, quantifying key acidity inducing factors could provide theoretical bases for combating soil acidification, and provide scientific and technological supports for red soil acidification remediation in China. 【Method】To achieve our objective, data of fertilizer rate, biomass (or yield) of main crops and trees, and above-ground nutrient contents reported within the experimental site were obtained from a large number of published literatures and statistical yearbooks. We quantified the acidity-inducing factors (nitrogen cycling process, BC (base cation) absorption and acid deposition) based on classical mass and charge balance. Relative contributions of the three key processes were used to illustrate the dominant factor of the soil acidification in uplands, paddy fields and woodlands. 【Result】For the whole county, nitrogen cycling process accounted for 66.5% (65.3% - 68.8%) of the total H + production, base absorption accounted for 33.0% (30.1%-34.4%), and acid deposition accounted for 0.5% (0.3% - 1.7%). Regardless of the land use patterns, nitrogen cycling process was the main source of H + production and main controlling factor of soil acidification. Among the three land use patterns, H + net production of upland was the highest (19.01 kmol·hm -2·a -1), followed by paddy field (16.5 kmol·hm -2·a -1), and woodland (3.2 kmol·hm -2·a -1) as the lowest. H + net production in dry farmland was about 6 times of woodland. H + net production of 6 main crop systems varies from 10.1 kmol·hm -2·a -1 to 30.0 kmol·hm -2·a -1, and followed the order: soybean>rape>peanut>rice>corn>sweet potato. Acidity production of the economic crops (rape, peanut and soybean) was generally higher than that of the grains (rice, corn, sweet potato); contribution rate of acidity-inducing of nitrogen cycling process among 6 main crop systems varied from 45.3% to 78.3%, contribution rate of acidity-inducing of base absorption varied from 21.4% to 54.2% . H + net production of 7 main woodland systems varied greatly from 1.96 kmol·hm -2·a -1 to 27.8 kmol·hm -2·a -1, and followed the order: citrus>chestnut>camellia oleifera abel>pine>fir>bamboo>slash pine. Acidity production of economic forest (citrus, chestnut and camellia oleifera abel) was generally higher than that of timber forest (pine, fir, bamboo and slash pine) ; contribution rate of acidity-inducing of nitrogen cycling process among 7 main woodland systems varied from 46.1% to 80.8%, and contribution rate of acidity-inducing of base absorption varied from 19.0% to 53.3% . The long-term field experiment combined with soil buffering curve technique was used to verify reliability of the calculation method of H + production. The simulated value of soil pH was positively correlated with the measured value significantly, with root mean square error (RMSE) of 0.15, while anastomosis degree between the two was high. 【Conclusion】 Nitrogen cycling process was the main controlling factor of red soil acidification in Qiyang County. The differences of total acidity production and contribution of acidity-inducing factors depended largely on land use patterns, crop types and tree species.

    Effects of Water and Nitrogen Regulation on Greenhouse Gas Emissions and Warming Potential in Vineyard Soil
    LIU Qiao,JI YanZhi,GUO YanJie,ZHANG LiJuan,ZHANG Jie,HAN Jian
    Scientia Agricultura Sinica. 2019, 52(8):  1413-1424.  doi:10.3864/j.issn.0578-1752.2019.08.011
    Abstract ( 377 )   HTML ( 8 )   PDF (475KB) ( 307 )   Save
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    【Objective】The objective of this paper was to explore the emission characteristics and warming potential of the greenhouse gases N2O, CO2 and CH4 from table grape vineyard soils under different water and nitrogen regulation, and to understand the contribution of water and nitrogen regulation to greenhouse gas emissions, thus screening out a more reasonable management model of water and nitrogen regulation, so as to provide scientific basis and technical reference for reducing greenhouse gas emissions from vineyards and promoting grape industry sustainable production. 【Method】From April 2017 to December 2017, Changli, the main grape producing area in Hebei Province, was selected as the experimental site and the table grape “Red globe” was used as the tested grape variety. A field microplot experiment was employed with four treatments, including traditional water and nitrogen, mobile water and fertilizer, optimized water and nitrogen, as well as optimized water and nitrogen + DMPP. The greenhouse gas emissions (N2O, CO2 and CH4) from the vineyard soil were monitored by using closed static chamber-gas chromatography, and then their comprehensive warming potential differences were compared. Final, the grape yields were measured. 【Result】N2O emission flux showed a single peak trend after fertilization, and the peak appeared on the 1-2 day after fertilization. Nitrogen fertilizer could significantly increase soil N2O emission flux. Compared with the traditional water and nitrogen treatment, nitrogen reduction and water control treatments could reduce the average N2O emission flux by 73.03%-88.19%, and their difference was significant (P<0.05). Optimized water and nitrogen + DMPP treatment could reduce the N2O emission flux by 50.08% on average under the condition of equal nitrogen, and the trend of CO2 emission flux was the same in all treatments, reaching the peak 2-3 days after fertilization, showing seasonal variation in the growth period. Nitrogen reduction and water control treatments could reduce CO2 emissions by 60.56%-62.13%. CH4 emission flux had no obvious change trend, but CH4 emission flux was positive or negative after fertilization. The traditional CH4 emission flux fluctuated greatly, ranging from -0.132 to 0.238 μg·m -2·h -1. There was no significant difference between nitrogen reduction and water control treatments (P>0.05). During the whole experiment period, the total N2O emissions of the treatments were in the order of traditional water and nitrogen, optimized water and nitrogen, mobile water and fertilizer and optimized water and nitrogen+DMPP, which were 3.90, 2.83, 2.76 and 2.65 kg·hm -2 with the emission coefficients were 0.58%-0.67%, respectively. Comparing with traditional water and nitrogen treatment, the nitrogen reduction and water control treatments (mobile water and fertilizer, optimized water and nitrogen and optimized water and nitrogen+DMPP ) could reduce the total N2O emissions by 27.56%-32.09%. The cumulative emissions of CO2 and CH4 were 3 816.05 kg·hm -2and 0.060 g·hm -2 in traditional water and nitrogen treatment, 3 387.33 kg·hm -2and -0.075 g·hm -2 in mobile water and fertilizer treatment, 3 410.95 kg·hm -2and -0.036 g·hm -2 in optimized water and nitrogen treatment, and 3 412.06 kg·hm -2 and -0.030 g·hm -2 in optimized water and nitrogen +DMPP treatment, respectively. Nitrogen reduction and water treatments could reduce the total cumulative CO2 emissions by 10.59%-11.23% and CH4 emissions by 150.23%-224.38%, respectively. Combining with the grape yield, the grape yield in nitrogen reduction and water control treatments was increased by 8.81% to 19.35% compared with traditional water-nitrogen treatment, and the largest increase was found under the optimized water and nitrogen + DMPP treatment, which was 9.69% and 2.25% higher than that under optimized water and nitrogen and mobile water and fertilizer treatment.【Conclusion】 Compared with the traditional water and nitrogen treatment, the cumulative emission of N2O, CO2 and CH4 in soils treated with optimized water and nitrogen + DMPP was decreased by 32.09%, 10.59% and 150.23%, respectively, and the total GWP was decreased by 12.82%, achieving greenhouse gas emission reduction in vineyards; at the same time, it could increase the grape yield by 19.35%, achieving a win-win situation for both economy and environment, which was evaluated as the best water and fertilizer regulation measures in this study.

    HORTICULTURE
    Soil Nutrient Status in Wuyi Tea Region and Its Effects on Tea Quality-Related Constituents
    ZHOU Zhi,LIU Yang,ZHANG LiMing,XU RuiNeng,SUN LiLi,LIAO Hong
    Scientia Agricultura Sinica. 2019, 52(8):  1425-1434.  doi:10.3864/j.issn.0578-1752.2019.08.012
    Abstract ( 513 )   HTML ( 32 )   PDF (4014KB) ( 697 )   Save
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    【Objective】 The objective of this research was to study the relationship between soil nutrient status and tea quality in Wuyi Tea Region, the main production area of Oolong tea, so as to provide theoretical basis for improving nutrient management in tea plantation and subsequent tea quality. 【Method】 Based on the soil survey data from Fujian Province in 2008, 68 tea plantations from three main tea production areas, including 12, 32 and 24 tea plantations from Tongmu, Rock tea and Zhou tea area, respectively, were randomly selected in Wuyi Tea Region in 2015. Soil samples from 0-20 cm top soil layer and one-tip-three-leaf tissue samples were collected from each tea plantation. Five soil nutrient indexes, including pH value, soil organic matter (SOM), alkaline nitrogen (AN), available phosphorus (AP) and available potassium (AK) concentrations in the top 0-20cm soils, and the concentrations of 6 secondary metabolites as quality factors in the fresh tea leaves (theanine, caffeine, rutin, ECG, EGCG and total catechins) were quantitatively measured by High Performance Liquid Chromatography (HPLC). 【Result】 Comparative analysis of soil nutrient indexes in 2008 and 2015 showed that soils throughout the Wuyi Tea Region were severely acidified, and those in a number of locations there were dramatic increased in AP over that time span. Among the three main tea production areas, soil nutrient status in Rock tea area was most significantly changed, which pH value, SOM and AN concentrations were reduced 0.65, 45.29% and 49.39%, respectively. While its AP was largely increased from 5.21 mg?kg -1 to 245.70 mg?kg -1 with over 40 times increase, indicating that excessive fertilization existed in the tea plantations of this region. Soil nutrient status significantly affected tea quality, with each quality factor being uniquely affected by particular soil nutrient indexes. The results from the marginal effect analysis revealed that the highest concentration of each secondary metabolite was associated with a corresponding suitable range for each soil nutrient index. Based on these associations, we proposed the suitable soil nutrient ranges for high-quality tea plantations in the Wuyi Tea Region were as follows: pH, 4.5-5.0; SOM, 20-40 g·kg -1; AN, 60-100 mg·kg -1; AP, 10-100 mg·kg -1; and AK, 100-150 mg·kg -1. 【Conclusion】Taken together, we suggested that the overall nutrient management in the areas of Rock tea and Zhou tea of Wuyi Tea Region were as follows: partially replace chemical fertilizers with organic fertilizers, properly supplement nitrogen and potassium, while strictly control phosphorus fertilization.

    FOOD SCIENCE AND ENGINEERING
    Flavor Variation of Flammulina velutipes in Polyethylene Film Packaging During the Cold Storage
    MA Ning,WANG ChaoFan,FANG DongLu,DING MengTing,YAO JiaLei,YANG WenJian,HU QiuHui
    Scientia Agricultura Sinica. 2019, 52(8):  1435-1448.  doi:10.3864/j.issn.0578-1752.2019.08.013
    Abstract ( 376 )   HTML ( 18 )   PDF (687KB) ( 248 )   Save
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    【Objective】 The investigation of flavor changes of Flammulina velutipes during the cold torage will provide scientific basis for preservation of F. velutipes. 【Method】 F. velutipes were packed with Polyethylene film and were stored at 4℃ and 90%RH for 15 days. Sampling was conducted every three days for analysis. The volatile components of F. velutipes were detected by headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (SPME-GC/MS) and electronic nose (E-NOSE). The non-volatile components of F. velutipes were determined by high performance liquid phase (HPLC), amino acid automatic analyzer and electric tongue (E-TONGUE). 【Result】 Monitoring the kinetics of flavor component in F. veluptipes during cold storage revealed that the main volatile components were aldehydes and alcohols. The contents of aldehydes and alcohols decreased at the beginning of postharvest storage, and then increased in the late storage stage (15 days). However, the mannitol and fructose content of F. velutipes were increased by 8.6% and 9.9%, respectively; during the initial storage (3 days) with a decrease in the middle (9 days) and end storage time (15 days). The citric acid content was decreased by 67.9% during storage. The reduction of succinic acid was observed in the first 3-day storage but increased by 17.94% in the middle of storage (9 days), finally decreased by 17.61% at the end of storage (15 days). The trends for the changes of 5'-AMP and 5'-IMP were similar to that of mannitol that the contents increased during the middle stages of storage (9 days) but dropped at the end of storage (15 days). The content of umami amino acid decreased at the initial stage of storage (3 days), while no significant difference was observed in the middle of storage (9 days). At the end of storage (15 days), the content of umami amino acid showed an upward trend. 【Conclusion】With the extended storage time, the profile of flavor changes in F. velutipes could be effectively fingerprinted, which would provide scientific basis for the quality control and the preservation of F. veluptipes.

    ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT
    Polymorphism of Rs319699771 Locus of Anti-Diarrhea MUC13 Gene in Suhuai Pig Population and Their Association with Economic Traits
    LIU ChenXi,WANG BinBin,PU Guang,ZHANG Qian,CAO Yang,WANG Huan,GAO Chen,NIU PeiPei,LI PingHua,HUANG RuiHua
    Scientia Agricultura Sinica. 2019, 52(8):  1449-1457.  doi:10.3864/j.issn.0578-1752.2019.08.014
    Abstract ( 391 )   HTML ( 27 )   PDF (469KB) ( 298 )   Save
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    【Background】 MUC13 gene is one of the main genes, which can regulate the infection of enterotoxigenic Escherichia coli F4ac, and cause diarrhea in piglets before weaning. The rs319699771 locus of the gene (G/A mutation) can accurately identify susceptible and resistant individuals, among which GG type is an anti-diarrhea genotype. The anti-diarrhea molecular breeding of Suhuai pig has been implemented by selecting GG genotype individuals, but whether the selection of GG genotype individuals will have adverse effects on other important economic traits, such as growth, carcass and meat quality, which is not yet clear. 【Objective】 The purpose of this study was to analyze the association between the locus and other economic traits to determine whether the molecular selection based on the rs319699771 locus of anti-diarrhea MUC13 gene would have adverse effects on other economic traits of Suhuai pig. 【Method】 In this experiment, 313 Suhuai fattening pigs weighting 87.61±0.54 kg were tested as experimental animals, and their carcass and meat quality traits were determined through slaughter. 261 Suhuai gilt pigs aging for 161.1±0.5 d were tested as experimental animals, and their growth and body size phenotype were also determined. At the same time, the corresponding ear tissue samples of Suhuai fattening pigs and gilt pigs were collected to extract tissue DNA. After Multiplex-PCR reactions, the polymorphism of the rs319699771 locus of MUC13 gene was detected in each pig. The association analysis between the polymorphism genotype of the rs319699771 locus of MUC13 gene and meat quality, carcass and growth traits were conducted by using the general linear model in SAS software. 【Result】 The results of polymorphism of rs319699771 locus of MUC13 gene showed that among the frequency of anti-diarrhea G allele of rs319699771 locus of MUC13 gene, the male and female pigs of the fattening pigs reached 0.695 and 0.634, respectively, and the anti-diarrhea GG genotype frequency was 0.467 and 0.373, respectively, in the male and female fattening pigs. While the locus G allele among the male and female pigs of the gilt reached 0.690 and 0.705, respectively, and the anti-diarrhea GG genotype frequency was 0.508 and 0.480, respectively, in the male and female gilt pigs. This indicated that the frequency of anti-diarrhea gene in Suhuai pig belonged to a higher level, and it could be feasible for the further improvement of anti-diarrhea GG frequency in the Suhuai pig through molecular breeding. The association analysis between polymorphism of rs319699771 locus of MUC13 gene and economic traits of Suhuai pig showed that there was no significant association between the locus’s polymorphism and carcass, meat quality traits in the fattening pigs (P>0.05), and it could be seen that increasing the breeding of the rs319699771 locus of MUC13 gene in Suhuai fattening pigs would not affect the carcass and meat quality traits. The locus’s polymorphism in the gilt extremely associated with the ham circumference index (P<0.01), and on average, the ham circumference of GG type individuals at this locus was about 1.46 cm longer than that of AG type individuals, and about 3 cm longer than that of AA type individuals. Also, the locus tended to associate with daily gain and terminal measurement weight (P<0.10), GG type individuals showed an upward trend compared with AA and AG type individuals. All of the three traits, GG genotype was a favorable genotype. The results showed that the breeding of the anti-diarrhea GG genotype of this locus could improve the growth traits of Suhuai gilt pigs. 【Conclusion】 Based on these results, the anti-diarrhea allele frequency of the rs319699771 locus of MUC13 gene in Suhuai pig was high, and the anti-diarrhea breeding was feasible, and the anti-diarrhea ability of Suhuai pig could be improved by selecting and remaining resistant homozygous GG genotype of rs319699771 locus of MUC13 gene and raising the frequency of the resistant homozygous genotype. At the same time, it could also realize the improvements of ham circumference and daily gain of Suhuai pig.

    Review of Measurement Technologies for Air Pollutants at Livestock and Poultry Farms
    WANG KaiYing,WU JieGang,ZHAO Xiaoyang
    Scientia Agricultura Sinica. 2019, 52(8):  1458-1474.  doi:10.3864/j.issn.0578-1752.2019.08.015
    Abstract ( 515 )   HTML ( 39 )   PDF (516KB) ( 758 )   Save
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    With the intensification of livestock breeding, the air quality problem of livestock farms caused by high density breeding is becoming more and more serious. Animal husbandry has become one of the important sources of air pollutants in China. Air emitted from most intensive livestock houses contains a large amount of pollutants, including ammonia, sulfides, particulate matters (PM), volatile organic compounds (VOCs), which not only poses a big threat to animals and workers in livestock farms, but also spreads to the surrounding environment resulting in air pollution. Scientific and applicable air pollutants measuring methods are the basis of monitoring and controlling air pollution in livestock and poultry farms. In this article, the detection methods of livestock farming related hazardous gases (e.g., NH3, H2S), greenhouse gases, particulate matters and odor were summarized. The detection methods of hazardous gases in livestock houses mainly include chemical analysis, semiconductor gas sensor detection, spectroscopic methodology and mass spectrometry. The wet-chemical method is cheap and can detect gases sensitively and accurately, while it cannot detect gases in real time, and the process is time-consuming and labor-intensive. The gas tube is cheap and easy to operate, but the deviation is great. Electrochemical sensor is of high sensitivity, moderate cost and can be used to detect gas concentration continuously, however, the devices are easy to age. Spectrum method and mass spectrometry can detect gas quickly and accurately, but it is not suitable for conventional air detection of productive livestock farming due to its high costs. In this paper, the detection methods of greenhouse gases (e.g., CH4, CO2) generated from animal intestinal fermentation and livestock environment were also summarized. It is hard to conclude an accurate detection of greenhouse gases in animal husbandry, because the concentrations of greenhouse gases in animal husbandry changes all the time (diurnal and seasonal) and are related to other factors including sampling points. No international common testing method and measurement standard are concluded till now, therefore, the research of greenhouse gases detection method and standard in animal husbandry should be carried out as soon as possible. The detection methods of particulate matters (PM) in livestock farms were reviewed from three aspects: physical, chemical and biological characteristics. PM contains complex components in livestock farms, therefore, it is highly needed to improve PM detection technology. Besides, the component analysis and sensory analysis of odorous substances in livestock farms were overviewed. The odor analysis of professional olfactory discernment personnel owns stronger subjectivity and costs higher than gas chromatography- mass spectrometry. While, using gas chromatography-mass spectrometry is unable to determine all gaseous organic compounds with one sample. Combining gas chromatography and dynamic olfactometer can be more efficient for comprehensive analysis of odor samples. In this article, detection methods and techniques of air pollutants in animal husbandry were comprehensively reviewed to provide a reference for the development of air pollutants detection technologies in livestock and poultry breeding in China.