Table of Content

01 November 2017, Volume 50 Issue 21

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Photosynthetic Characteristics and Its Physiological Basis of Super High-Yielding Hybrid Rice Liangyoupeijiu

Lü ChuanGen, LI Xia, CHEN GuoXiang

Scientia Agricultura Sinica. 2017, 50(21):  4055-4070.  doi:10.3864/j.issn.0578-1752.2017.21.001

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Two-line super hybrid rice Liangyoupeijiu (Peiai64S/9311) is a landmark of China’s super rice project and proud of the largest planting area in the last fifteen years. Compared with its parent 9311 and the large-scale planted hybrid rice Shanyou 63, Liangyoupeijiu has obvious yield superiority and competitive advantage. The most important biological basis is its good photosynthetic characteristics which results in higher biomass. Studying its photosynthetic characteristics could help to understand the photosynthetic growth of high yield hybrid rice, and improve the light efficiency by exploratory breeding technology, and provide a theoretical basis and technical approaches for super high-yielding rice breeding. After summarization of the various relevant studies, it was found that, in comparison with 9311 and Shanyou 63, Liangyoupeijiu shows higher light absorption, transmission and transformation efficiency and carbon assimilation which result in obvious advantages in Pn and other photosynthetic characters. The characters are: showing adaptation or tolerance of higher or lower light intensity even UV-B enhance, and stronger resistance to aging; with various strategies to resist lower or higher temperature, showing stronger adaptation or tolerance of damaged temperatures; showing less damage under water stress; photosynthetic rate increased with nitrogen levels, and had advantages of photosynthetic capacity both in lower and higher nitrogen; photosynthesis increased by higher atmospheric CO₂ concentration, and tolerance stronger on soil pH, salt stress and so on. Those excellent photosynthetic properties have their physiological basis. Its chlorophyll content is higher, and declined later and slowly, which results in a higher chlorophyll content in its late growing stage. It has a higher Rubisco initial carboxylation activity, total carboxylation activity and activation rate. The expression of C₄ photosynthetic pathways is up-regulated by its higher activity of C₄ enzymes, and caused both lower photosynthetic compensation point of light intensity and CO₂ concentration, and a lower photorespiratory activity as well, which shows obvious C₄ photosynthetic characteristics. The activities of antioxidase such as SOD and POD in its leaves are higher, which eliminate the endogenous O₂^.- and H₂O₂, so that it shows more stronger resistance to membrane lipid peroxidation. The authors consider that hybrid rice breeding with high-yielding should pay more attention to "three excellent traits" by combining with genetic trait of strong heterosis, morphological trait of plant ideotype, and functional trait of fine photosynthetic performance. Exploring the relationship among C₄ photosynthetic pathways, high photosynthesis and grain yield of Liangyoupeijiu, and studying photosynthetic characteristics of more other new types of hybrid with super high yield are both very meaningful to deepen the understanding of photosynthetic growth of such high-yielding hybrid rice, and explore the targeted technology to promote the C₄ photosynthesis function for further improving of the photosynthetic production and grain yield in future hybrid rice production.

Tapetal Programmed Cell Death, Antioxidant Response and Oxidative Stress in Wheat Anthers Associated with D²-type Cytoplasmic Male-sterility

LIU ZiHan, SHI XiaoYi, YAN PengJiao, DUAN Yang, GENG XingXia, YE JiaLi, LI Sha, YANG XueTong, ZHANG GaoMing, JIA YuLin, ZHANG LingLi, SONG XiYue

Scientia Agricultura Sinica. 2017, 50(21):  4071-4086.  doi:10.3864/j.issn.0578-1752.2017.21.002

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【Objective】D² type cytoplasm is the considerable source of cytoplasm of cytoplasmic male sterility (CMS). It is of great value to further study the cytological characteristics and mechanism of physiology and molecules in heterologous cytoplasmic male sterility (CMS) wheat for the heterosis utilization. 【Method】To elucidate the abortive cytological characteristics of D²-type cytoplasmic male-sterility, the phenotypes of anthers, microspores, tapetum were analyzed by scanning electron microscope, DAPI staining, and paraffin section, as well as detectingtapetal apoptotic DNA fragmentation by DNA ladder. To further clarify that the D²-type cytoplasmic male-sterility is associated with abnormal tapetal programmed cell death and reactive oxygen species metabolism, the physiological properties, including the contents of reactive oxygen species (ROS), activities of ROS-scavenging enzymes (superoxide dismutase (SOD), peroxidase, catalase (CAT), ascorbate peroxidase (APX), and glutathione peroxidase (GPX)) and nonenzymatic antioxidants (ascorbic acid (AsA) and glutathione (GSH)), and the expression levels of antioxidant-associated genes from three D²-types of cytoplasmic male sterile lines (Va87B1-706A, C₆87B1-706A, Ju87B1-706A) and the maintainer 706B of wheat were compared.【Result】Compared with the maintainer line, at the trinucleate stage, the inner epidermal cells of D²-type cytoplasmic male-sterility accumulated less and more unconsolidated Ubisch bodies and the epidermis of microspores were extremely scabrous. The abortive pivotal period of pollen was initiated at the later uninucleate stage, which the delayed degradation of tapetum and abnormal structure of the microspore were related to the serious oxidative stress, disorganized activities of antioxidant enzymes, down-regulated contents of non-enzymatic antioxidants and significant difference of the expression level of some related enzyme genes in the three D²-types of cytoplasmic male sterile lines. Additionally, the expression level of SOD and APX genes was approximated to the activities of enzymes. However, there existed a distinct difference between the expression level of CAT gene and the activities of enzymes resulting from multiple-gene encoding CAT gene.【Conclusion】It was deduced that delay of start-up of the tapetal PCD (Programmed Cell Death) results from excessive ROS accumulation, and then destroyed the balance of the antioxidant defense system, and eventually led to the abortion of D²-types of cytoplasmic male sterile lines. Therefore, in order to further explore the abortive mechanism of cytoplasmic male sterility (CMS) wheat, this study provided an important theoretical basis and application significance.

Genetic Dissection of Grain Filling Related Traits Based on a High-Density Map in Maize

GAO Xing, LI YongXiang, YANG MingTao, LI BeiBei, LI ChunHui, SONG YanChun, ZHANG DengFeng, WANG TianYu, LI Yu, SHI YunSu

Scientia Agricultura Sinica. 2017, 50(21):  4087-4099.  doi:10.3864/j.issn.0578-1752.2017.21.003

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【Objective】The grain filling rate and grain filling duration are major determinants of grain yield. Examination of the contributions of growth period and grain filling related traits to kernel weight, and study of quantitative trait loci (QTL) for grain filling rate and duration to dissect the genetic basis of grain filling are helpful to the practice for the breeding of high yield in maize.【Method】To identify QTL for grain filling rate and duration, a recombinant inbred line (RIL) population including 172 families was developed from the cross between Huangzaosi (HZS) and Lv28 which are foundation parents used in maize breeding of China. Firstly, the differences between the Logistic and the Richards models for the fitting of maize grain filling process were compared and the parameters of the grain filling were calculated. Secondly, the differences between HZS and Lv28 in grain filling characteristic were compared. Correlation analysis and regression analysis were applied to elucidate the relationship among growth period and grain filling related traits and the contribution to hundred-kernel weight (HKW). Thirdly, the approach of genotyping-by-sequencing (GBS) was used to detect polymorphic SNP markers between the parents and among the RILs. Finally, the inclusive composite interval mapping (ICIM) was used to identify QTL of growth period and grain filling related traits.【Result】The genetic map was constructed with 1 471 filtered SNP markers, the total length was 1 471 cM with the average length 1 cM. The grain filling process tended to be a slow-fast-slow pattern and could be divided into three phases: the lag phase, the effective grain filling phase and the maturation drying phase. The use of model fitting of the grain filling process could reflect the dynamic changes of grain filling, but the Richards model and the Logistic model were different in simulating the grain filling dynamic process. The r² measure of predicted values obtained by the Richards model with phenotypic values was higher than that by the Logistic model. The grain filling characteristics showed significant differences between the two parents, since the grain filling rate of HZS was 1.28 times higher than that of Lv28, while the grain filling duration of Lv28 was 1.07 times longer than that of HZS. Correlation analysis showed that the grain filling related traits and HKW reached a significant positive correlation except for the grain filling duration in the lag phase. Regression analysis showed that the grain filling rate and the grain filling duration could explain 57.50% and 30.00% of the phenotypic variation of HKW, respectively. The effective grain filling phase played an important role in the formation of HKW. A total of 26 QTL were detected for grain filling related traits, 3 QTL were detected for HKW and 14 QTL were detected for growth period related traits under the single environment, which were distributed on the chromosomes 1, 2, 3, 4, 5, 6, 8, 9 and 10. The LOD values ranged from 3.27 to 9.05, and the range of phenotypic variation explained was 5.97%-21.16%. Under the joint environments, the QTL controlling the grain filling related traits were located at the same or similar positions of chromosomes and formed some QTL clustering regions on bin 1.05, bin 2.03, bin 4.05, bin 4.06, bin 7.04 and bin 9.04. Importantly, some QTL for grain filling rate in different grain filling phases were detected to be clustered on the region of 48.24 cM-135.72 cM on chromosome 4 and the region of 110.10 cM-114.73 cM on chromosome 9. 【Conclusion】 Maize grain filling dynamic process could be simulated well with the Richards model. The grain filling rate and duration of the effective grain filling phase played an important role in the formation of HKW. The inheritance of grain filling related traits were strongly influenced by the environments, so some QTL could be detected only in one environment. Under the joint environments, some QTL related to grain filling rate were detected on bin 4.05 and bin 9.04.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Quality Consistency of Soft Wheat Yangmai 13 Under Different Nitrogen Application

GAO DeRong, SONG GuiHua, ZHANG Xiao, ZHANG BoQiao, LI Man, JIANG Wei, WU SuLan

Scientia Agricultura Sinica. 2017, 50(21):  4100-4106.  doi:10.3864/j.issn.0578-1752.2017.21.004

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【Objective】The objectives of this study were to evaluate the effect of nitrogen application rate and regime on dough properties and end-use quality of soft wheat Yangmai 13, and to provide information for the production and breeding of soft wheat. 【Method】 Field experiments were conducted over two cropping seasons (2012-2014) in Lixiahe Regional Institute of Agricultural Sciences of Jiangsu. Yangmai 13 was planted in a randomized complete block design with three replications at four different nitrogen treatments (A1: 120 kg·hm^-2, A2: 180 kg·hm^-2, A3: 240 kg·hm^-2, A4: 300 kg·hm^-2). The nitrogen application ratio of A1, A2 and A3 treatments in seedling stage, tillering stage and jointing stage was 7:1:2, and A4 treatment was based on 5:1:4. Quality parameters, including protein content, wet gluten content, SDS sedimentation value, farinograph parameters, alveograph parameters, SRC values and cookie qualities, were investigated to understand the effect of nitrogen fertilizer on the quality of Yangmai 13. 【Result】The results indicated that protein content, wet gluten content and dough development time were influenced significantly by nitrogen amount and regime. However, slight effect was observed for SDS sedimentation value, stability, water absorption, P value, L value, P/L, W value, SRC value, and cookie quality parameters except for cookie thickness. Increasing the amount of N application increased protein content and wet gluten content. Grain yields were found to increase with increasing nitrogen fertilizer, while differences among A2, A3 and A4 were non-significant. The factor of year had no significant effect on protein content, wet gluten content, water absorption, P value or cookie diameter, while other quality parameters were strongly influenced by year. There were no significant interactions between nitrogen fertilizer and year for most quality parameters, except for protein content, wet gluten content, dough development time, cookie thickness and fracturability. 【Conclusion】 Nitrogen application amount and regime have no significant influence on most physical-chemical quality parameters, including SDS sedimentation value, SRC value, farinograph parameters and alveograph parameters, and cookie processing quality. Therefore, Yangmai 13 could be planted as a stable quality soft wheat. The results also suggest that it is possible to breed soft wheat varieties with good and stable quality to N application.

Effects of Water and Phosphorus Coupling on Root Growth, Biomass Allocation and Yield of Quinoa

PANG ChunHua, ZHANG ZiWei, ZHANG YongQing

Scientia Agricultura Sinica. 2017, 50(21):  4107-4117.  doi:10.3864/j.issn.0578-1752.2017.21.005

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【Objective】In arid regions, agricultural plantation is mainly restricted by water and fertilizer. To explore a optimal water/phosphorus ratio and provide a theoretical basis for the high yield of quinoa in arid land. In this study, the effects of water and phosphorus coupling on root growth, biomass allocation and high yield of quinoa (Chenopodium quinoa Willd) were studied. 【Method】 Guinoa was taken as an experimental material and pot-culturing experiment were carried out at 3 levels of water irrigation (W1, W2 and W3 represented 35%-45%, 55%-65% and 75%-85% soil moisture content) and 4 levels of phosphorus fertilizer (P₂O₅ application rates of P0, P1, P2, P3 were 0, 0.1, 0.2 and 0.4 g·kg^-1), and root morphological and physiological indexes, biomass allocation and yield under the conditions of 12 different water and phosphorus coupling treatments were compared. 【Result】 At the same water irrigation level, the root parameters (root area, total root length, maximum root length, root diameter and root volume) were the highest under P2 treatment. At the same phosphorus level, the maximum root length and total root length reached the maximum under W2 treatment, and root area showed W2P0＞W3P0 and W2P1＞W3P1 under low phosphorus treatment (P0, P1). Under high phosphorus treatment (P2, P3) root area showed W2P2＜W3P2 andW2P3＜W3P3. Root diameter and root volume increased with the increasing of water content. Under the severe drought stress (W1) with P1 treatment, root activity were highest; but it was the highest under other water irrigation levels with P2 treatment. POD and SOD activities were highest and MDA was lowest under P2 (0.2 g P₂O₅·kg^-1) treatment across all irrigations; The appropriate phosphorus fertilizer (P2) significantly reduced the soluble sugar and proline content. The appropriate ratio of water and phosphorus (W3P2, W3P1) was beneficial for  biomass of stem and leaf weight, and yield of quinoa, while the weight of root and panicle were the highest under W2P3 treatment. High water irrigation benefited for biomass allocation between stems and leaves; Low water irrigation was beneficial for biomass allocation between root and panicle at 3 water levels, P2(0.2 g P₂O₅·kg^-1) treatment was suitable for formation of apical spike; Low phosphorus promoted the number of branch and spike, 1000-grain weight and grain weight, while they were specific under high phosphorus conditions, and at various levels of phosphorus with W3 treatment gain weight reached the maximum, respectively. 【Conclusion】 The suitable phosphorus supplement (P2, 0.2 g P₂O₅·kg^-1) improved root activity, root growth, increased area of the root contacting and soil, strengthened antioxidant ability of root, and therefore enhanced the drought resistance of quinoa. and the optimal ratio of water and phosphorus (W3P2) was suitable for biomass accumulation and high yield of quinoa.

PLANT PROTECTION

Effects of Predator-Induced Stress from Harmonia axyridis on the Development and Stress Protein Gene Expression of Helicoverpa armigera

YAN Shuo, XIONG XiaoFei, CHU YanNa, LI Zhen, WU PengXiang, YANG QingPo, CUI WeiNa, XU JinTao5, XU LiXia, ZHANG QingWen, LIU XiaoXia

Scientia Agricultura Sinica. 2017, 50(21):  4118-4128.  doi:10.3864/j.issn.0578-1752.2017.21.006

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【Objective】 The objective of this study is to determine the effects of predator-induced stress from Harmonia axyridis with various feeding resources on the development and metamorphose of Helicoverpa armigera, illustrating that whether He. armigera can perceive and classify the predation risk, and show the trade-off characteristic between the development and metamorphose, and to determine the effects of long-term and short-term stress on the stress protein gene expression of He. armigera, illustrating that whether the predator-induced stress from Ha. axyridis can induce the physiological reactions of He. armigera at molecular level. 【Method】The development (larval and pupal duration, female and male longevity, and total longevity) and metamorphose (pupae weight, pupation rate, fail eclosion rate, and wrinkled-wing rate) indicators of He. armigera were observed and recorded under the predator-induced stress from Ha. axyridis with 7 kinds of feeding resources (hungry treatment, shrimp egg treatment, cotton bollworm larva treatment, cotton bollworm egg treatment, aphid treatment, CK treatment with aphid, and CK treatment). Long-term (first instar larvae to 3-day-old moth) and short-term (15 min to 6 h) stress treatments were set up, and the changes of stress protein genes (heat shock protein genes) Hsp70 and Hsp90, heat shock cognate protein gene Hsc70 expression were determined under the predator-induced stress by quantitative real-time PCR (qRT-PCR). 【Result】Under the predator-induced stress from Ha. axyridis, larval and pupal duration, female and male longevity, and total longevity of He. armigera shortened, pupal weight and pupation rate decreased, and wrinkled-wing rate increased significantly, whereas the fail eclosion rate was not influenced by the predator-induced stress. Under the predator-induced stress from Ha. axyridis with various food sources, the larval duration of He. armigera was the shortest when predators consumed aphids, the pupal duration was the shortest when predators consumed cotton bollworm eggs, and the total longevity was the shortest when predators consumed shrimp eggs, whereas the female and male longevity were not influenced by the diets of Ha. axyridis. The wrinkled-wing rate of He. armigera was the highest when predators consumed cotton bollworm eggs, whereas the pupal weight, pupation rate, and fail eclosion rate were not influenced by the diets of Ha. axyridis. Stress protein genes Hsp70 and Hsp90 were up-regulated after short-term stress (Hsp70: 30 min to 3 h; Hsp90: 15 min, 1.5 h, 2 h and 6 h), whereas heat shock cognate protein gene Hsc70 was up-regulated after long-term stress (the stages of 5th instar larvae, prepupa, male pupae and male moth). 【Conclusion】Under the long-term stress from Ha. axyridis, all developmental stages of He. armigera shortened, and the development of He. armigera became faster to avoid the predation risk. The developmental acceleration might disturb the metamorphose of He. armigera to some degree, leading the smaller pupal weight, lower pupation rate, and higher wrinkled-wing rate, which was according with the trade-off hypothesis. The sensitiveness of He. armigera to predator-induced stress were different among various diets of Ha. axyridis, and He. armigera might be able to classify the potential predation risk, however this ability showed a degree of uncertainty. The predator-induced stress from Ha. axyridis could induce the physiological reactions of He. armigera at molecular level, leading the higher expression levels of stress protein genes. Hsp70 and Hsp90 expressions were more affected by the short-term stress, whereas Hsc70 expression was more affected by long-term stress.

The feasibility of using graminaceous weeds as a functional plant for controlling rice leaffolder (Cnaphalocrocis medinalis)

ZHENG XuSong, TIAN JunCe, YANG YaJun, ZHU PingYang, LI Kuan, XU HongXing, Lü ZhongXian

Scientia Agricultura Sinica. 2017, 50(21):  4129-4137.  doi:10.3864/j.issn.0578-1752.2017.21.007

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【Objective】Increasing plant diversity is an effective way for sustainable pest control through trapping pests, protecting natural enemies. Host plants of rice leaffolder (Cnaphalocrocis medinalis) include rice and some graminaceous plants. The objective of this study is to develop a new strategy of C. medinalis control based on the preference of C. medinalis to graminaceous plants and rice plants. 【Method】Seven common graminaceous weeds (Leersia sayanuka, Paspalum distichum, Digitaria sanguinalis, Leersia hexandra, Leptochloa chinensis, Echinochloa crusgalli, and Eleusine indica) around rice fields were selected as the potential hosts in the experiments. These weeds were planted in pots and were placed into cages with rice plants, C. medinalis adults were also introduced into the cages, then thehabitat and oviposition preference of C. medinalis to graminaceous weeds and rice, and the biological fitness of C. medinalis on graminaceous weeds were studied. Furthermore, the effects of graminaceous weeds, which were planted on bund as 1 m width, on the distribution and oviposition of C. medinalis and egg parasitoids were evaluated in rice fields. Meanwhile, population density of C. medinalis in bund planted with different graminaceous weeds and that in the site with 1, 5 and 10 m from bund were recorded by chasing moth method. Number of C. medinalis eggs, parasitized eggs and parasitism rate on different graminaceous weeds in the field were also investigated. 【Result】C. medinalis adults prefer to the habitat of L. sayanuka and D. sanguinalis, and do not prefer P. distichum. There was no significant difference between L. hexandra, E. indica, L. sayanuka, L. chinensis and rice. C. medinalis adults prefer to lay eggs on rice compared with L. sayanuka, E. crusgalli, E. indica, L. chinensis, P. distichum and L. hexandra. L. sayanuka significantly attracted C. medinalis adult to lay more eggs than rice. No significant difference on oviposition preference was found between D. sanguinalis and rice. C. medinalis could complete their generation on all seven tested graminaceous weeds. However, significant difference on development time was detected among treatments. Lowest development time was found on L. sayanuka, followed with P. distichum. There was no significant difference on development time among L. hexandra, L. chinensis, E. indica, D. sanguinalis, E. crusgalli and rice. Plant hosts significantly impact the pupal weight. Female pupal weight from D. sanguinalis, male pupal weight from D. sanguinalis and L. chinensis were significantly lighter than those from the other host plants. The emergence rates of C. medinalis from different host plants varied largely, which from L. sayanuka, L. hexandra, E. indica, E. crusgalli, and rice were ranged from 42.17%-51.31%; but which fromL. chinensis and P. distichum were as low as 11.76% and 13.29%, respectively. Female rate of C. medinalis reared on rice and E. crusgalli was significantly higher than those reared on the other host plants. Field experiments indicated that graminaceous weeds could attract more C. medinalis adults than rice, except E. indica and E. crusgalli. There were significant differences on the number of C. medinalis eggs on different graminaceous weeds, which was ranged from 2.92-3.92 per tiller on L. sayanuka, L. hexandra, E. crusgalli and L. chinensis. The mean number of C. medinalis eggs on P. distichum and D. sanguinalis was 2.16 and 1.72, respectively; and lowest eggs on E. indica. The parasitism rate of C. medinalis eggs by egg parasitoids was varied significantly among different graminaceous weeds, ranged from 21.90%-55.61%. The mean number of parasitized C. medinalis eggs was 1.47 and 1.42 per tiller on L. sayanuka and L. hexandra,respectively, which was highest than those on the other graminaceous weeds. 【Conclusion】The results imply that L. sayanuka has the most potential to be used as a functional plant for controlling C. medinalis. The results could provide a theoretical foundation for developing new green C. medinalis management technology.

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

Influence Factor Analysis of Farmland Soil Heavy Metal Based on the Geographical Detector

LI Yu, HAN Ping, REN Dong, LUO Na, WANG JiHua

Scientia Agricultura Sinica. 2017, 50(21):  4138-4148.  doi:10.3864/j.issn.0578-1752.2017.21.008

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【Objective】To study the correlation between soil heavy metals and influencing factors and heavy metals in different soils, as well as to provide a more comprehensive auxiliary variable for soil heavy metal spatial prediction model.【Method】The spatial distributions of heavy metals Pb, Cd, As, Cr and Hg in five soils in five towns of Xiangtan County, Hunan Province were analyzed by using the geophysical model and the spatial interpolation technique. The correlation and interaction of spatial distributions of heavy metals and 6 factors, as well as that of five heavy metals, were studied.【Result】The results showed that Gross Domestic Product (GDP), the average temperature and relative humidity had a greater explanatory power to the five kinds of soil heavy metals to (P_{D, H} values are above 0.5). Soil pH, soil type, elevation and soil heavy metals were less explanatory (P_{D, H} values below 0.3). The soil type had the lowest explanatory power to five kinds of soil heavy metals (P_{D, H} values were below 0.1). Among the five soil heavy metals, Cr has the strongest explanatory effect on Cd (P_{D, H} = 0.95), and As is the least (P_{D, H} = 0.20). The effects of average temperature, relative humidity and GDP on soil heavy metals were significantly higher than those of other Influence factors, while the difference of explanatory power between other influence factors was not significant. There are mutually reinforcing or non-linear enhancement effects between the Influence factors and between the five heavy metal elements. 【Conclusion】The spatial distribution of heavy metals in soil is the result of the interaction of multiple influence factors. Based on the geographical exploration model, the Influence factors, such as GDP, average precipitation, average temperature and relative humidity, have strong explanatory power for the spatial distribution of heavy metals of research area in soils. These influence factors can be used as the soil heavy metal space in the study area predictive model of the auxiliary variable. Geographic detector model can provide a more comprehensive analysis of various influence factors, and provide an effective basis for the establishment of soil heavy metal spatial prediction model.

Accumulation Characteristics and Risk Assessment of Heavy Metals in Typical Greenhouse Vegetable Bases

XU Li, LU AnXiang, TIAN XiaoQin, HE HongJu, YIN JingWei

Scientia Agricultura Sinica. 2017, 50(21):  4149-4158.  doi:10.3864/j.issn.0578-1752.2017.21.009

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【Objective】The objective of this paper is to study accumulation characteristics and health risk of heavy metals in greenhouse vegetable base of Beijing, to determine the migration rule of heavy metals from soil to vegetable, and to provide reference basis for vegetable quality safety and optimization of vegetable structure in Beijing. 【Method】 148 soil samples and 96 vegetable samples were collected from 9 typical greenhouse vegetable bases in Beijing, the statistical characteristics of As, Cd, Cr, Hg and Pb in soil and vegetable samples and their transfer factors from soil to vegetable system were analyzed. Combined with the EPA (US) recommended health risk assessment model, the health risks of adults and children as a result of vegetable intake were evaluated. 【Result】 The mean concentrations of As, Cd, Cr, Hg and Pb in soils were 9.43, 0.18, 64.4, 0.11 and 21.6 mg·kg^-1, respectively. The mean concentrations of As, Cd, Cr, Hg and Pb in vegetables were 0.0478, 0.0391, 0.2785, 0.0014 and 0.0454 mg·kg^-1, respectively. On the whole, the migration ability of heavy metals in greenhouse vegetable bases were decreased in the order of Cd＞Hg＞Cr＞As＞Pb. Target hazard quotients caused by heavy metals were decreased in the order of Cd＞Pb＞Hg＞As＞Cr. The target hazard quotient of single heavy metal in all vegetable samples was less than 1, which indicated that there was no obvious negative effect of single heavy metal. 【Conclusion】 The accumulation of heavy metals (As, Cd, Cr and Hg) in the soil samples from the greenhouse vegetable bases were found, health risks should be emphasized.

Simultaneous Determination of Multi Herbicides Residues in Vegetables by Dispersive Solid Phase Extraction and Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

PING Hua, LI Yang, LI BingRu, HE ZhaoYing, LIU JiPei, MA ZhiHong

Scientia Agricultura Sinica. 2017, 50(21):  4159-4169.  doi:10.3864/j.issn.0578-1752.2017.21.010

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【Objective】 To study the purification efficiency of different amount of absorption on the recovery of the herbicides, the ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with dispersive solid phase extraction method was developed for the determination of 24 herbicides in vegetables．【Method】 The vegetable samples were extracted with acetonitrile and purified by primary-secondary amine (PSA) with different dosage of 0.05 g, 0.10 g and 0.20 g. UPLC-MS/MS method was performed with electrospray ionization (ESI) source under the multiple reaction monitoring (MRM) mode．【Result】0.1% formic acid water-acetonitrile was chosen as mobile phase in gradient elution, and the peaks of all the compounds were sharp with high sensitivity under this conditions. 24 kinds of herbicides were detected by UPLC-MS/MS within 10 min. Adding 0.10 g and 0.20 g PSA, the recovery rate of acifluorfen, 2,4-Dicholrophenoxyacetic acid, flumetsulam, thifensulfuron methyl, tribenuron methyl, and chlorimuron ethyl was decreased significantly, and the rest of the herbicides recovery did not change significantly, ranging from 75.5% to 114.7%. When adding 0.05 g PSA, 24 herbicides recovery rate was 65.6%-114.7%. Standards concentration ranged from 1.0 µg·L^-1 to 250 µg·L^-1 had a good linear relationship, and the correlation coefficient was 0.9946-0.9998. Limits of detection of 24 herbicides in vegetables ranged from 1.2 to 160.8 µg·kg^-1, and limit of quantitative between 4.0 and 536.0 µg·kg^-1, which can satisfy the requirement of quantitative analysis. The mixed standard solution of 24 herbicides were spiked in the blank vegetable samples at three levels 50, 100, 200 µg·kg^-1, and the recovery of 24 kinds of herbicides at the rate of 66.5%-116%, the relative standard deviation between 2.3%-9.7%, which can meet the requirements. Using the established determination method, 38 vegetable samples were detected, and only atrazine was detected in all vegetable samples with the detection rate of 57.9% and the detection range from not detected to 0.84 µg·kg^-1. The other remaining 23 kinds of herbicides were not detected.【Conclusion】The UPLC-MS/MS method is simple, rapid, solvent saving and sensitive, which is suitable for the determination of herbicides in a large number of vegetable samples.

HORTICULTURE

Effects of Green LED Light on the Growth and Quality of Lettuce

CHEN XiaoLi, YANG QiChang, ZHANG Xin, MA TaiGuang, GUO WenZhong, XUE XuZhang

Scientia Agricultura Sinica. 2017, 50(21):  4170-4177.  doi:10.3864/j.issn.0578-1752.2017.21.011

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【Objective】 To explore the better mode of green LED light supply in plant factories, the effects of different modes of green light on the growth and nutrient quality of lettuce were analyzed.【Method】Lettuce was grown in the fully artificial light plant factory, where adjustable white and green LED panels were used as the sole light source for lettuce growth. White light (W) provided at 8:00-20:00 (160 μmol·m^-2·s^-1) was regarded as the basal light for normal growth of lettuce, and green light (G) at different intensity (30, 60, and 90 μmol·m^-2·s^-1) was supplied as the supplemental light. Meanwhile, overlapping mode (O) and non-overlapping mode (N) of the basal white light and the green light were set by regulating the green light time. Treatments were recorded as W, WG₃₀O, WG₆₀O, WG₉₀O, WG₃₀N and WG₉₀N, and the green light period for all treatments was 6 h. 【Result】 All treatments were observed to enhance the shoot fresh weight of lettuce compared with the control except treatment of WG₉₀N, 30 μmol·m^-2·s^-1 low intensity of supplemental light was more beneficial for the growth and biomass accumulation of lettuce. Supplemental green light promoted the accumulation of soluble sugar, crude protein and vitamin C at different levels, and decreased the nitrate content at the same time. Among which, the soluble sugar, crude protein and Vc contents increased with the increase of green light intensity. Green light depended on the exist of basal white light on the biomass accumulation of lettuce, but the green light didn’t show the dependent on the basal white light on the process of the soluble sugar and vitamin C. 【Conclusion】 The effects of green light supply are relevant to the light intensity as well as the supply modes. Green lighting strategy can be established and adjusted according to the production purpose in the actual production.

Molecular Cloning and Expression Analysis of Cytokinins Responsive Gene MdMYB308 in Red Flesh Apple

WANG YiCheng, WANG Nan, XU HaiFeng, ZHANG ZongYing, JIANG ShengHui, ZHANG Jing, QU ChangZhi, CHEN XueSen

Scientia Agricultura Sinica. 2017, 50(21):  4178-4185.  doi:10.3864/j.issn.0578-1752.2017.21.012

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【Objective】Cytokinin is an important hormone in the regulation of anthocyanin synthesis in plants. To develop the theory and technology for red flesh apple breeding, the function in the cytokinin regulating anthocyanin metabolism of MYB transcription factor gene MdMYB308 in ‘Zihong No.3’ from Malus sieversii f. neidzwetzkyana F₁ population was studied. 【Method】The callus induced from the leaves of ‘Zihong No.3’ apple was used as materials. The MdMYB308 in ‘Zihong No.3’ was cloned and its bioinformation was analyzed. The expression level of MdMYB308 and anthocyanin biosynthesis related genes in callus which grown on different concentrations of 6-BA was studied by the qRT-PCR. Meanwhile, the interaction between MdbHLH3 and MdMYB308 was verified by yeast two-hybrid system and bimolecular fluorescence complementation assay. 【Result】 The full length of MdMYB308 in ‘Zihong No.3’ was cloned, and the gene was 768 bp which encoded 255 amino acids. It was predicted that the molecular mass of this protein was 28.37 kD, and pI was 8.94. A phylogenetic tree indicated that MdMYB308, AtMYBL2, FaMYB1 and AtMYB4 are located in the same evolutionary branch. The aligned protein sequences revealed that MdMYB308 contain the EAR motif. Furthermore, the content of anthocyanin rose as 6-BA concentration increased as well. The transcript levels of anthocyanin structural genes (MdCHS, MdDFR, MdUFGT) and transcription factors (MdMYB10, MdbHLH3) were significantly higher in callus grown on 1 mg?L^-1 6-BA compared with 6-BA-deprived callus. In contrast, the expression of MdMYB308 was inhibited. The results of yeast two hybrid experiments and bimolecular fluorescence complementation assays showed that the MdMYB308 could interact with MdbHLH3.【Conclusion】Cytokinin (6-BA) could promote anthocyanin accumulation by down-regulating the expression of MdMYB308 which may destroy the combination of MdMYB308 and MdbHLH3.

FOOD SCIENCE AND ENGINEERING

An Electrochemical Aptasensor for Detection of Salmonella Based on Composite Nanomaterial and Enzymatic Recycling for Amplification

XU LianYing, WANG BiNi, ZHANG FuXin

Scientia Agricultura Sinica. 2017, 50(21):  4186-4195.  doi:10.3864/j.issn.0578-1752.2017.21.013

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【Objective】 Salmonella is an important detection target of pathogeny bacteria in food. In order to overcome the shortcomings of traditional Salmonella detection methods, a novel assay of electrochemical aptasensor for quantitative detection of Salmonella with better practicability was established.【Method】Reduced Graphene Oxide (rGO) solution and toluidine blue (Tb) solution were mixed together to obtain the Tb-rGO nanocomposite, and then the Tb-rGO nanocomposite was dispersed in gold nanoparticles (AuNPs) colloidal solution to obtain the AuNPs-Tb-rGO nanocomposite. Then, the as-prepared AuNPs-Tb-rGO nanocomposite was incubated with amino-DNA to obtain the DNA-nanocomposite (S₁-AuNPs-Tb-rGO). The complementary strands of the aptamers of Salmonella (S₂) were attached to the surface of gold electrode by Au-S-bond, and then the electrode surface was blocked with HT. Subsequently, the aptamers of Salmonella (Apt) were dripped onto the modified electrode to make Aptbind with S₂. The modified electrode was immersed into the mixture containing Salmonella and exonuclease I (Exo I). In terms of the characteristics of Exo I that could amplify electrical signals and the aptamers that could exclusively bind with Salmonella, the aptamers were taken away from S₂ circularly. Then, the S₁-AuNPs-Tb-rGO composite was attached to the surface of electrode by the hybridization of S₁ and S₂. Finally, the conditions of the incubation time in bacteria liquid, the Exo I concentration and the S₁-AuNPs-Tb-rGo composite concentration were optimized and the electrical signals of the electrode surface was monitored to construct the aptasensor. This electrochemical aptasensor was used to test Escherichia coli, Staphylococcus aureus, Shigella, Listeria monocytogenes and Enterobacter sakazakii to ensure the electrochemical aptasensor’s specificity. The electrochemical aptasensor was used to detect 6×10²-6×10⁶ cfu/mL Salmonella to ensure the electrochemical aptasensor’s sensitivity. Then this electrochemical aptasensor was used to detect the goat milk to evaluate the practical use of electrochemical aptasensor.【Result】The optimization of the electrochemical aptasensor incubation time in bacterial liquid, the Exo I concentration and the S₁-AuNPs-Tb-rGo composite concentration were studied in detail, and the optimal conditions were 1 h, 0.6 U?µL^-1 and 200 nmol?L^-1. When Salmonellas were tested existent, they had specific binding with Apt and the S₁-AuNPs-Tb-rGo composite was attached to the electrode surface. So the linear sweep voltammetry curve of the electrochemical aptasensor showed a rise of oxidation peak. The developed aptasensor was specific to Salmonella and did not react with non-target bacteria. The electrochemical aptasensor detected the Salmonella target at a titer higher than 200 cfu/mL. A good recovery of Salmonella in the range of 91.6%-106.3% was obtained in goat milk by electrochemical aptasensor assays developed.【Conclusion】This electrochemical aptasensor can detect Salmonella with a easy operation, a wide linear range, a high sensitivity and a low cost, which provide good application prospects in the field of rapid quantitative detection of salmonella.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Effect of Leucine and Phenylalanine Supplementation on Growth Performance and Serum Metabolites of Holstein Male Calves

YANG XinJian, CAO YangChun, ZHENG Chen, LIU Kai, GUO Long, CAI ChuanJiang, LIU BaoLong, YAO JunHu

Scientia Agricultura Sinica. 2017, 50(21):  4196-4204.  doi:10.3864/j.issn.0578-1752.2017.21.014

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【Objective】The objective of the study was to know the effect of leucine and phenylalanine supplementation on growth performance and serum metabolites of Holstein male calves. The study could provide a reference for the high efficient use of leucine and phenylalanine on dairy cows.【Method】A total of 20 Holstein male calves (birth weight (38±3) kg) were randomly assigned into treatment groups of control, L (1.435 g·L^-1 leucine), P (0.725 g·L^-1 phenylalanine), M (1.435 g·L^-1 leucine and 0.725 g·L^-1 phenylalanine), and each treatment added alanine as isonitrogenous diet. The experiment lasted for eight weeks, one week for adaption and seven weeks for feeding experiment. The calves were fed individually. Within one hour after birth, each calf was fed 4.0 L of colostrum. During the first week, the calves were fed their assigned milk twice daily, 3.0 L each. For adaption, the amino acid supplementation was increased 20% daily until it reached 100% of the target supplementation on the sixth day after birth. From week 2 to 3, the calves were fed their assigned milk twice a day, 3.5 L each, while from week 4 to 8, the volume of the milk fed increased to 4 L each time. From the third week after birth of the calves, a quantitative supply of the starter feed was provided, with free access to the drinking water. Body weight, withers height, body length, heart girth, heart girth/body length, body length/withers height were recorded at the 1, 3, 5, 7, and 8 week old, respectively. At the same time, 10 mL of blood was drawn from the jugular vein and placed at a tilted angle for serum precipitation. The serum samples were used to determine the amino acids, glucose, serum urea nitrogen, insulin, and cholecysto-kinin, respectively. Data were analyzed using the GLM procedure of IBM SPSS Statistics V22.0 software, differences among treatments were compared using LSD’s multiple range tests.【Result】Average daily gain in M group was significantly lower than control, L and P groups (P＜0.05). Compared with control group, other three groups significantly increased the final body length (P＜0.05). L and M treatments tended to increase the final wither height (P＜0.10), while P treatment significantly decreased the final heart girth (P＜0.05). Compared with control group, L and M treatments significantly increased the heart girth/body length (P＜0.05), whereas no difference in initial body weight, body length, wither height, heart girth, heart girth/body length, body length/withers height, final body weight and final body length/withers height was found among treatments (P＞0.05). L treatment significantly increased the concentrations of histidine, leucine, threonine, glycine, and total essential amino acids (P＜0.05), tended to increase the concentrations of aspartate and total amino acids (P＜0.10), while significantly decreased the concentrations of alanine and glycine (P＜0.05). P treatment significantly increased the concentrations of phenylalanine, glutamate and glycine (P＜0.05). Compared with L and M groups, control group significantly increased the concentrations of serum alanine (P＜0.05). For serum concentrations of amino acids, no significant interactions between L and P treatments were detected in our study (P＜0.05). L treatment significantly increased the concentrations of glucose (P＜0.05), while P treatment significantly decreased the concentrations of glucose (P＜0.05). For serum concentrations of glucose, significant interactions between L and P treatments were detected in our study (P＜0.05). Serum urea nitrogen in P group was significantly higher than other three groups (P＜0.05), whereas no difference in concentration of insulin and cholecysto-kinin were found among treatments (P＞0.05).【Conclusion】In conclusion, leucine supplementation had a better effect than that of phenylalanine on the serum concentration of essential amino acids, glucose, heart girth/body length and average daily gain of Holstein male calves, whereas negative interactions existed between the two kinds of supplementation.

Effect of Folic Acid on Lipid Metabolism Associated Gene Expression in Primarily Cultured Chickens Hepatocytes

LIU YanLi, DANG YanNa, DUAN YuLan, YANG XiaoJun

Scientia Agricultura Sinica. 2017, 50(21):  4205-4211.  doi:10.3864/j.issn.0578-1752.2017.21.015

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【Objective】Abdominal fat deposition is a universal phenomenon at present in poultry industry. Different from mammals, 90%-95% of lipid metabolism in poultry occurs in the liver. Previous studies have demonstrated folic acid and IGF2 were involved in animals lipid metabolism. The study was conducted to establish the method for primary culture of chicken hepatocytes and investigate the effects of folic acid on IGF2 and genes expression associated with lipid metabolism in primary chicken hepatocytes, further providing a basis for exploring lipid metabolism of poultry in vitro. 【Method】Chicken hepatocytes were isolated by collagenase digestion combined with purification of chicken lymphocyte separation fluid, and evaluated by PAS staining, respectively. After 36 h culture of hepatocytes, cells were treated with different concentration of folic acid for 12 h. Hepatocytes proliferation and injury was detected by MTT method and LDH activity in culture medium respectively. Cells were collected to get total RNA for gene expression analysis by RT-PCR. Later, correlation analysis was carried out between IGF2 and lipid metabolism related genes expression. Regression analysis was performed between folic acid concentration and genes expression.【Result】The results showed that hepatocytes were isolated with high purity. Folic acid didn’t affect cell proliferation and cellular LDH activity (P＞0.05).When compared with the control group (1 mg·L^-1 folic acid), 10, 15 and 20 mg·L^-1 folic acid significantly reduced IGF2 expression in chicken hepatocytes (P＜0.05), and same phenomenon was observed in FAS and ACC expression. However, folic acid had no effects on CPT-1 and PPARα expression (P＞0.05) . In addition, FAS and ACC expression had positive correlation with IGF2 mRNA level in chicken hepatocytes (P＜0.05). There existed linear and quadratic regression between genes expression and folic acid concentration (P＜0.05). 【Conclusion】 In conclusion, folic acid could reduce gene expression associated with fatty acid synthesis in chicken hepatocytes. In addition, FAS and ACC mRNA level might have positive connection with IGF2 expression. Optimum treatment dose of folic acid was 15 mg·L^-1 in this study.

RESEARCH NOTES

Genome-Wide Association Study of Grain Peroxidase Activity in Common Wheat

SHI Jia, ZHAI ShengNan, LIU JinDong, WEI JingXin, BAI Lu, GAO WenWei, WEN WeiE, HE ZhongHu, XIA XianChun, GENG HongWei

Scientia Agricultura Sinica. 2017, 50(21):  4212-4227.  doi:10.3864/j.issn.0578-1752.2017.21.016

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【Objective】 Peroxidase (POD) activity has browning and bleaching effects on the color of flour and flour-based products during processing and storage. Identification of associated loci and candidate genes for grain POD activity is important for molecular marker-assisted selection (MAS) in wheat quality breeding. 【Method】 In the present study, the POD activities were surveyed with 151 and 82 Chinese bread wheat cultivars from Yellow & Huai Winter Wheat Region (YHRVWWR) and Northern China Plain Winter Wheat Region (NWWR), respectively, and each set of cultivars was planted in four environments. A genome-wide association study (GWAS) was performed using the mixed linear model (MLM) based on 18 189 and 18 417 high-quality SNP markers from 90K SNP array for two sets of cultivars, respectively. 【Result】The POD activity of the tested materials showed extensive phenotypic variation and diversity. The variation coefficient of YHRVWWR was 15.4%-21.8%, the heritability was 0.79, and the variation coefficient of NWWR was 15.0%-19.9%, the heritability was 0.82. The POD activity of the materials in different environments showed a significant correlation, and the correlation coefficients were 0.46-0.89 (P＜0.0001) and 0.50-0.87 (P＜0.0001) in YHRVWWR and NWWR, respectively. The polymorphic information content of value was between 0.09-0.38, and the minimum allele frequency was between 0.05-0.5. The population structure analysis showed that the two natural populations in YHRVWWR and NWWR were simple and could be divided into three subgroups. In the YHRVWWR cultivars, 20 loci were found to be associated with POD activity (P＜0.001), which were located on chromosomes 1A, 2A, 2B, 2D, 3A, 3B, 3D, 4A, 4B, 5A, 5B, 6A, 6D and 7A, and each explained 7.8%-13.3% of phenotypic variation. In the NWWR cultivars, 20 loci showed significant association with POD activity (P＜0.001), which were located on chromosomes 1A, 1B, 1D, 2A, 2B, 2D, 3A, 3B, 4B, 6A, 6B, 7A, 7B and 7D, explaining 14.4%-23.2% of phenotypic variation. Two loci were detected in both the YHRVWWR and NWWR cultivars, and the associated SNPs could be used to develop STARP (Semi-thermal asymmetric reverse PCR) or CAPS markers. The regression analysis showed that the POD activity of wheat grain was higher with the increasing number of favorable alleles. Meanwhile, three candidate genes PMM-D1, PER40,and F775_31640 were scanned, encoding phosphomannomutase, horseradish peroxidasesand alkyl hydro peroxide reductase, respectively. 【Conclusion】The genetic diversity of the two natural populations in YHRVWWR and NWWR are rich in genetic structure and were suitable for genome-wide association analysis. Twenty POD activity loci were found in 2 natural populations, respectively, and three candidate genes were detected. Regression analysis showed that the more favorable alleles variation, the higher the POD activity.

The Influence of Brassinosteroid on the Light Catching, CO2 Fixation and the Translocation of Organic Compounds in Maize Leaves

ZANG JinPing, ZHAO AiJia, ZHAO YaLin, YAN QingDi, FENG JiaJia, ZHANG HaiLi, WANG FengRu, DONG JinGao

Scientia Agricultura Sinica. 2017, 50(21):  4228-4234.  doi:10.3864/j.issn.0578-1752.2017.21.017

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【Objective】The objective of this study is to clear the influence and the molecular mechanism of brassinosteroid on the photosynthetic characteristics of action in maize (Zea mays L.) leaves, and then to provide the theoretical basis and technical references for efficient using of brassinosteroid in maize field. 【Method】We sprayed 100 nmol·L^-1 brassinosteroid on maize of 8 leaves period, then observed and analyzed the chloroplast structure, starch accumulation, chlorophyll content, phosphoenol plyruvate carboxylase (PEPC), photosynthetic rate, the activity of sucrose phosphate synthase (SPS) and sucrose synthase (SS) in leaves. 【Result】After 15 days from brassinosteroid treatment, compared with the control treatment, the net photosynthetic rate increased by 32.6%. At the same time, the accumulation of starch grains in the chloroplast was increased significantly. Compared with the control, the chlorophyll content of the leaves treated with brassinosteroid increased by 28.57%. The above results showed that brassinosteroid treatment can improve the light-harvesting ability of maize leaves. PEPC is an enzyme that catalyzes the fixation of CO₂ in C₄ plants. The results showed that brassinosteroid treatment can improve the activity of PEPC in maize leaves, and the activity of PEPC increased by 14.52% compared with the control. This showed that brassinosteroid treatment can improve the ability of fixing CO₂ of maize leaves. Transportation of photosynthetic products is an important factor to determine yield. Through the ultrastructural observation of the phloem tissue cells of maize leaves, it was found that the cell inclusions in the phloem conducting tissue increased under brassinosteroid treatment. Sucrose is the main transportation form of photosynthetic products. Sucrose synthase and sucrose phosphate synthase are key enzymes in sucrose synthesis, and their activity can reflect the transport capacity and strength of photosynthetic products input to grain. In this study, the activity of sucrose synthase and sucrose phosphate synthase increased by 28.26% and 30.20%, respectively, under brassinosteroid treatment. The results showed that brassinosteroid treatment could improve the output ability of maize photosynthetic products.【Conclusion】 Brassinosteroid can raise the light energy utilization of maize blades by raising the photosynthetic pigment content, can enhance the capacity of maize leaf CO₂ fixation by raising the PEP carboxylase activity, and can promote the transformation and transportation of organic matter in maize leaf through raising the activity of sucrose synthase and sucrose phosphatase.