Table of Content

01 December 2017, Volume 50 Issue 23

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Guidance of the SPECIAL FOCUS "Breeding of Foxtail Millet and Common Millet":: Current Breeding Situation of Foxtail Millet and Common Millet in China as Revealed by Exploitation of 15 Years Regional  Adaptation Test Data

DIAO XianMin, CHENG RuHong

Scientia Agricultura Sinica. 2017, 50(23):  4469-4474.  doi:10.3864/j.issn.0578-1752.2017.23.001

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Originated from China, foxtail millet (Setaria italica Beauv.) and common millet (Panicum miliaceum L.) are staple cereals in Northern China, which are of great importance in dry-land agriculture. Organized by the National Agricultural Technology Extension Center, newly developed foxtail millet and common millet cultivars were subjected to evaluate their grain yield, environmental adaptation and other agricultural characteristics performance in different locations of their corresponding ecological regions from 2001 to 2015, which is named Regional Adaptation Test (RAT for short). The 15 years RAT of foxtail millet and common millet cultivars were carried out on different ecological groups, and there were 8 to 15 testing locations of the same of similar environmental condition in each group, such as Northern China Plain region of summer-sowing foxtail millet, early mature region of Northwest China spring-sowing region and Northeast China spring-sowing region. The well-organized data of the 15 years test is a real exhibition of performance of grain yield and other characters of the newly developed foxtail and common millet cultivars in this period. By exploitation of the data, six papers were published in this issue of Scientia Agricultura Sinica and a brief introduction and comment was made in this paper, so as help readers have a much deeper insight on those papers. The future breeding targets of foxtail and common millet were also discussed in the paper.

The Alterations of Foxtail Millet Breeding in North China Summer-Sowing Region from 2001 to 2015

ZHANG Ting, SHI ZhiGang, WANG GenPing, GAO Xiang, XIA XueYan, YANG WeiHong, ZHANG XiRui, TIAN XiaoJian, CHENG RuHong, DIAO XianMin

Scientia Agricultura Sinica. 2017, 50(23):  4475-4485.  doi:10.3864/j.issn.0578-1752.2017.23.002

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【Objective】By analyzing the yield and agronomic traits alterations of foxtail millet cultivars tested in North China summer-sowing region from the latest 15 years, the change trend in millet breeding level was elucidated to provide references for the future development of foxtail millet cultivars.【Method】The main agronomic traits data of foxtail millet cultivars in North China summer-sowing region from 2001 to 2015 was used to reveal the changing rule of foxtail millet breeding. Correlation analysis between climatic factors and agronomic traits of foxtail millet cultivars tested from 2001 to 2015 was conducted. The types of foxtail millet cultivars were classified.【Result】The agronomic traits of the cultivars altered significantly during 2001 to 2015. As time lapse, yield, growth duration, plant height, panicle length, panicle weight and panicle grain weight increased, while 1000-grain weight decreased slightly, and panicle numbers per hectare decreased. The change trend of the main agronomic traits of the 51 foxtail millet cultivars from year 2001 to 2015 was in accordance with all tested cultivars. Variance analysis showed that yield, growth duration, plant height, panicle length, 1000-grain weight and panicle numbers per hectare were significantly different among the 51 foxtail millet cultivars, while there was no significant difference in panicle weight, panicle grain weight and percentage of grain weight per panicle. The climate trend was warm and humid in North China summer-sowing region. The yield had a higher positive correlation with the growth duration, panicle weight and panicle grain weight, but had a higher negative correlation with the lowest temperature and precipitation. 85.17% of yield variation was determined by lowest temperature, highest temperature, precipitation, growth duration, panicle grain weight and percentage of grain weight per panicle. The lowest temperature had a negative effect on yield, while the highest temperature had a positive effect on yield. The foxtail millet breeding level was progressed from 2001 to 2015, and the types of foxtail millet cultivars were abundant with more herbicide resistance cultivars and high quality cultivars, showing that the main breeding direction was simplified cultivation and quality. However, there were 26 foxtail millet cultivars with the parental source of Jigu19, Yugu1 and Jigu25. The percent was up to 57.8%, showing that the parents used in millet breeding were in a narrow scope.【Conclusion】The yield of foxtail millet cultivars in North China summer-sowing region from 2001 to 2015 was increased somewhat, the types of foxtail millet cultivars were abundant, and the foxtail millet breeding level was progressed to some extent. However, the yield difference was mainly caused by weather fluctuation. In the future, the types of parent should be immensely enriched. And the types of millet cultivars with higher grain weight and higher percentage of grain weight per panicle should be cultivated for adapting climate change and improving yield.

Evaluation of Foxtail Millet Cultivars Developed in the Middle and Late-Maturing Spring-Sowing Region in Northwest China in 2005-2015

ZHANG AiYing, GUO ErHu, DIAO XianMin, FAN HuiPing, LI YuHui, WANG LiXia, GUO HongLiang, CHENG LiPing, Wu YinSheng

Scientia Agricultura Sinica. 2017, 50(23):  4486-4495.  doi:10.3864/j.issn.0578-1752.2017.23.003

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【Objective】 The objective of this study is to analyze the changes of agronomic traits, grain yield, stress resistance and quality traits of newly developed foxtail millet cultivars in the middle and late-maturing spring-sowing region in northwest China in 2005-2015­, and provide scientific data for new cultivar development and genetic resource enhancement.【Method】 Based on the data of middle and late-maturing spring-sowing foxtail millet lines tested in the regional adaptation tests from 2005 to 2015 in northwest China, main agronomic traits and yield traits were analyzed and compared. Diseases resistances and quality traits of 30 foxtail millet cultivars released were further analyzed in this paper. 【Result】The grain yield potential of the newly developed foxtail millet cultivars in 2005-2015 showed a trend of increase from the data analyzed. Great difference of agronomic characteristics of tested lines and released cultivars among experimental years were identified, which implied that climate difference among years might have great impact on foxtail millet characters. The data showed a clear increasing trend in characteristics such as plant height, panicle weight, panicle grain weight and ratio of grain on a panicle of the newly developed cultivars, while the growth duration of those varieties became shorter than before. Coefficient correlation analyses of the agronomic traits indicated that grain yield of cultivars is positively correlated with panicle weight, grain weight per panicle and number of panicles per hectare and negatively related with the length of growth duration. The commonly occurred diseases in this foxtail millet growing region included leaf rust, blast, sheath blight, red leaf disease and downy-mildew in the past ten years and among which the latter two are the main ones. All the newly developed cultivars showed their increase in the abilities of diseases resistance. Most newly developed cultivars were characterized with yellow kernel, and the most adapted cultivars were usually characterized with a relatively lower content of crude protein and fatty acids, but a relatively higher lysine content. Different types of cultivars including waxy starch, high protein content, herbicide resistant were developed in the past ten years which provided firm foundation for foxtail millet industry in this region. 【Conclusion】The yield potential of newly developed foxtail millet cultivars in the middle and late maturing region of northwest China in the past 11 years was improved. Different types of cultivars were developed so as to meet foxtail millet industry in this region. The main method of foxtail millet breeding in this region is pedigree selection after hybridization between cultivars, which should be improved with other methods such as molecular marker assistant selection.

Research Progress and Major Traits of Foxtail Millet Cultivars Developed in the Early-Mature Spring-Sowing Region in the Past 15 Years

ZHANG AiYing, DIAO XianMin, GUO ErHu, FAN HuiPing, WANG LiXia, LI YuHui, CHENG LiPing, WU YinSheng, ZHANG Li

Scientia Agricultura Sinica. 2017, 50(23):  4496-4506.  doi:10.3864/j.issn.0578-1752.2017.23.004

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【Objective】The objective of this study is to analyze the changes of agronomic traits, yield and resistance characters of foxtail millet cultivars released in the early-mature spring-sowing region in China in the past 15 years, the progress and breeding direction of foxtail millet was discussed, which provided scientific evidence for new cultivars development in the future.【Method】 Based on the data obtained from early-mature spring-sowing foxtail millet cultivars that participated in the regional adaptation test from 2001 to 2015 in Northwest China, agronomic traits and grain yield traits were analyzed and compared. 【Result】There were 85 newly developed foxtail millet lines participated in the regional adaptation test from 2001 to 2015, among which 18 lines that meet the national standard of new cultivar were authorized to release. Among the 12 conventional cultivars and 6 hybrid cultivars that released, only three of them have the grain yield potential that is over 10% higher than the control. Great difference was identified among the average grain yield of lines that participated in the test between different years, but it shows a potential of increase of grain yield with the time schedule. There is no significant difference among the annual means of panicle length and 1000-grain weight of the tested and released foxtail millet lines and the range of panicle length and 1000-grain weight were 23-27 cm and 3-3.4 g respectively, while significant differences were identified between the annual means of plant height, panicle weight and panicle grain weight, which showed a gradual increase trend with the time schedule. Among the 18 cultivars released, the growth duration of 13 of them is longer than the control cultivars, and all those cultivars were characterized with taller plant height, heavier panicle weight and panicle grain weight, which imply that much more attention was paid on the selection of single plant characters in the breeding efforts. Co-relation coefficient analyses indicated that 81.43% of the grain yield variation was decided by growth duration, plant height, panicle weight, panicle grain weight and the rate of grain per panicle, and grain yield was significantly related with panicle weight and panicle grain weight. Diseases and drought resistance related characters of those lines participated in the test were also recorded which indicated that the resistant ability of stresses of released lines among the past 15 years in the early mature foxtail millet breeding became stronger. 【Conclusion】 There is a growth tendency of grain yield in the newly developed early mature foxtail millet cultivars in 2001-2015, the main characters that contributing to grain yield include panicle weight, panicle grain weight, plant height and growth duration. Considering the quick development of machinery cultivation, efforts of developing short plant height, strong logging resistance, big panicle with higher net grain rate should be paid much more attention.

Evaluation of Foxtail Millet Cultivars Developed in Northeast China Spring-Sowing Region in Recent Years

LI ZhiJiang, MA JinFeng, LI YanDong, LI XiangYu, DIAO XianMin, ZHANG Ting

Scientia Agricultura Sinica. 2017, 50(23):  4507-4516.  doi:10.3864/j.issn.0578-1752.2017.23.005

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【Objective】 The objective of this study is to analyze the agronomic traits, grain yield and other important characters of newly developed foxtail millet cultivars in the Northeast China in 2005-2015?, and provide scientific data for new cultivar breeding and germplasm utility.【Method】Based on the data of foxtail millets lines tested in the regional adaptation test of Northeast China from 2005 to 2015, main agronomic traits and yield traits were analyzed and compared with DPS software. 【Result】There were sixty cultivars tested in the regional adaptation test, and 18 cultivars were released according to the foxtail millet registration standard of the Chinese Agricultural Technology Extension Center in recent years. The grain yield potential of the newly developed foxtail millet cultivars showed variation according to the data analysis in 2005-2015, and it was influenced by climate greatly. The average yield of cultivars released in 2010 was higher than that of 2005 by 4.6%, and the average yield of cultivars released in 2015 was higher than that of 2005 and 2010 by 12.62% and 7.66% respectively. On the whole, the grain yield of cultivars released had a trend of increase. There was certain difference in the range of yield increase by the type of cultivars. The yield of Jiugu 23, Jiugu 14 and Longgu 31 was over 10% higher than that of the control cultivar. The result of correlation coefficient and stepwise regression analysis of the agronomic traits indicated that grain yield of cultivars is positively correlated with panicle weight and grain weight per panicle. Stepwise regression analysis indicated that 90.98% of the grain yield variation was decided by growth duration, plant height, panicle weight, the rate of grain per panicle, 1000-grain weight and the number of panicles per hectare. Jiugu 23 was the most prominent cultivar in the Northeast Spring-Sowing Foxtail Millet Region with a relative low plant height of 115 cm, which represent the direction of millet breeding. 【Conclusion】The yield potential of newly developed foxtail millet cultivars in the Northeast China Spring-Sowing Region in recent years was someway improved, which show a trend of yield increase year by year. The panicle weight and grain weight per panicle were the most important traits for grain yield, while the yield was also affected by climate and cultivation condition. The panicle weight and grain weight per panicle should be paid more attention during the breeding progress, and related traits such as plant height, growth duration and biotic/abiotic resistance affected the yield of foxtail millet as well. The plant height and panicle length should be reduced for cultivars adapting to simplified cultivation technology. Cultivars that have the characters of resistant to herbicide should be developed to satisfy the adjustment of Chinese crop farming system to reduce labor investment.

Changes of Yield and Traits of Broomcorn Millet Cultivars in China Based on the Data from National Cultivars Regional Adaptation Test

YANG Pu, Rabia Begum Panhwar, LI Jing, GAO JinFeng, GAO XiaoLi, WANG PengKe, FENG BaiLi

Scientia Agricultura Sinica. 2017, 50(23):  4517-4529.  doi:10.3864/j.issn.0578-1752.2017.23.006

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【Objective】 The changes in yield and agronomic traits of broomcorn millet cultivars were analyzed in the national cultivars regional adaptation tests made in recent 17 years. The progress of broomcorn millet improvement and the capacity of the breeding institutions in China were investigated, aimed to provide information for further genetic improvement of broomcorn millet in China. 【Method】 The multivariate regression analysis, correlation analysis and cluster analysis were employed. The phenotypical variation of the broomcorn millet cultivars in the national regional adaptation tests were analyzed from the year 1998 to 2014. The differences in traits among the cultivars that bred by different breeding institutions were compared. 【Result】 The traits altered significantly among the years from 1998 to 2014 for broomcorn millet cultivars. Yield and grain weight per plant of non-waxy millet, and the yield, grain weight and panicle length of waxy millet constantly increased over the years. In the past 17 years, the yield of non-waxy and waxy millet increased by 50% and 21%, respectively. The grain weight per plant was increased by 90% and 7%, respectively. Panicle length was increased by 19% and 29%, respectively. There were no significant alteration in growing duration, plant height, node number and grain weight. Seventeen breeding institutions from Heilongjiang, Jilin, Liaoning, Inner Mongolia, Hebei, Shanxi, Shaanxi, Gansu and Ningxia provided 65 cultivars in total for the national cultivar regional adaptation test. Of which, seven institutions provided both non-waxy and waxy cultivars, two institutions provided only the non-waxy cultivars, and eight institutions provided only the waxy cultivars. The breeding institutions from Inner Mongolia, Gansu and Ningxia contributed 55.4% of the cultivars in total. In the latest 6 years, the cultivars from these 3 regions showed excellent performance, thus the breeding institutions in these regions are the backbone of broomcorn millet breeding in China. Multivariate regression analysis on yield and agronomic traits showed that the growing duration, node number and grain weight per plant contributed 82.8% of the yield variation for the non-waxy millet, whereas in the waxy millet, the node number and grain weight per plant together determined 78.6% of the yield variation. The correlation analysis showed that in both the non-waxy and waxy millet, panicle length and grain weight per plant were significantly correlated with yield per plant, however, the correlation among other traits were different between non-waxy millet and waxy millet. The cluster analysis showed that the non-waxy cultivars were grouped into 2 categories, whereas it could be divided into 3 categories for the waxy cultivars. 【Conclusion】 The yield of non-waxy and waxy millet in China were increased steadily from 1998 to 2014, which indicated that the breeding ability of the institutions in China has increased. However, the breeding method is relatively simple and the diversity of the breeding method is low. Broomcorn millet breeders ignored the importance of the yield related traits and quality traits. As for the broomcorn millet breeding direction in the future, the cultivars with high yield, high quality, good palatability, good cooking quality, high resistant starch content, strong resistance to shattering and good adaptability for mechanized cultivation should be developed. Multiple breeding methods including the traditional breeding methods such as hybrid breeding, distant hybridization, mutation breeding, doubled haploid and polyploidy breeding technology, introducing the methodology of genome research and molecular breeding, and combining traditional breeding with modern biotechnology are the most important strategy for improving the breeding technology of broomcorn millet.

Comprehensive Evaluation of Agronomic, Yield and Quality Traits of Broomcorn Millet (Panicum miliaceum L.) Cultivars

YANG QingHua, QIU Jun, LI Hai, YANG TianYu, CHENG BingWen, ZHAO Min, LIU GuoQing, GAO XiaoLi, FENG BaiLi

Scientia Agricultura Sinica. 2017, 50(23):  4530-4544.  doi:10.3864/j.issn.0578-1752.2017.23.007

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【Objective】 In order to analyze the problems existed in broomcorn millet improvement and put forward solutions, the agronomic, yield and quality traits of non waxy and waxy broomcorn millet cultivars were evaluated. This would also provide theoretical supports for the breeding and development of broomcorn millet production. 【Method】The experimental materials were 22 non waxy broomcorn millet and 18 waxy broomcorn millet cultivars developed in recent years. The agronomic traits (growth duration, plant height, nodes, grain color, panicle color, and panicle shape) and yield traits (1000-grain weight, panicle weight and panicle size) were collected in field trials. The yellow pigment (n-butanol extraction), crude fat content (Soxhlet extraction method), amylose and amylopectin content (dual wavelength method) and protein content (Kjeldahl method) were obtained in laboratory.【Result】The average yield of non waxy broomcorn millet cultivars was 3 465.5 kg·hm^-2 (2 976.0-3 915.0 kg·hm^-2), and that of waxy broomcorn millet cultivars was 3 163.4 kg·hm^-2 (2 575.5-4 002.0 kg·hm^-2). The correlation analysis of agronomic traits showed that the correlation coefficient between yield and growth duration, 1000-grain weight, panicle size and panicle weight reached the significant level. The results showed that the growth duration, plant height, nodes, 1000-grain-weight, panicle weight, panicle size and yield of broomcorn millet cultivars were within a small range. The average yellow pigment of non waxy broomcorn millet cultivars was 2.7 mg·kg^-1 (1.7-3.3 mg·kg^-1), and that of waxy broomcorn millet cultivars was 2.4 mg·kg^-1 (2.1-2.9 mg·kg^-1). The average crude fat content of non waxy broomcorn millet cultivars was 3.6% (1.7%-5.6%), and that of waxy broomcorn millet cultivars was 4.1% (2.7%-5.5%). There was no significant difference between non waxy and waxy broomcorn millet in yellow pigment and crude fat content. The average amylose content of non waxy broomcorn millet cultivars was 32.22% (11.31%-38.67%), while the average amylopectin content was 35.01% (21.43%-64.02%). The average total starch content of non waxy broomcorn millet cultivars was 67.23% (58.59%-77.87%). The average ratio of amylose to amylopectin of non waxy broomcorn millet cultivars was 1.00 (0.18-1.73). The average amylose content of waxy broomcorn millet cultivars was 3.69% (2.24%-5.55%), while the average amylopectin content was 57.37% (49.40%-68.01%). The average total starch content of waxy broomcorn millet cultivars was 61.06% (54.18%-72.11%). The average ratio of amylose to amylopectin of waxy broomcorn millet cultivars was 0.07 (0.05-0.11). Among them, the content of amylose was higher in Longmi 5 and Longmi 8, and the content of amylose was moderate in Ningmi 17 and Yumi 2, and the content of amylose was low in Jinshu 9 and Yanshu 7. The average protein content of non waxy broomcorn millet cultivars was 11.13% (9.64%-13.26%), and that of waxy broomcorn millet cultivars was 13.72% (12.10%-15.72%). The protein content of waxy broomcorn millet was higher than that of non waxy broomcorn millet.【Conclusion】In recent years, variations in a number of agronomic and yield traits of broomcorn millet cultivars released was within a small range and the type of broomcorn millet cultivars was unitary, which could not meet the production and market demand. It is necessary to combine the traditional breeding method with the new molecular breeding technology in mining, utilizing and innovating the excellent millet resources. The direction of improvement of millet cultivars is to develop new cultivars with excellent traits, strong resistance to biotic and abiotic stress, wide adaptability, high yield and good quality.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Effects of Gibberellin on Migration and Colonization of Rhizobia and Seedling Growth of Alfalfa

MIAO YangYang, SHI ShangLi, KANG WenJuan

Scientia Agricultura Sinica. 2017, 50(23):  4545-4557.  doi:10.3864/j.issn.0578-1752.2017.23.008

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【Objective】The objectives of this study are to determine the effect of gibberellin on the migration and the colonization dynamics of the two fluorescent tagged rhizobia in Gannong No.5 alfalfa (Medicago sativa L., Gannong No.5) tissues, and also to determine the effects of the combination treatments on alfalfa seedlings growth, so as to the results will not only enhance the colonization ability especially in reproductive tissues, but also provide benefits for alfalfa cultivation.【Method】The effect of gibberellin on the rhizobia growth (OD₆₀₀) were detected at 1, 3, 5, 7 and 9 d after the day of adding gibberellin; the migration and colonization of rhizobia in alfalfa tissues of 15, 30, 45 and 60 day, and seedling growth were also investigated by drenching root with the inoculant of rhizobiaEnsifer meliloti 12531f (12531f) and Ensifer meliloti LZgn5f (gn5f), which were added with 0.5, 1, 10 and 100 mg·L^-1 gibberellin, respectively.【Result】The results showed that, under 10 mg·L^-1 and 1 mg L^-1 gibberellin level, 12531f and gn5f could grow better than other levels, but did not have significant effects. The two fluorescent tagged rhizobia could migrate to the aerial tissues and well colonize lower stems, upper stems and upper leaves by adding 10 mg·L^-1gibberellin into 12531f treatment and added 1 mg·L^-1 into gn5f treatment, respectively. The fluorescent tagged rhizobia could not be detected in the control (sterile distilled water) treatment. Inoculating 12531f and gn5f alone increased the leaf chlorophyll content, while the addition of gibberellin inhibited the leaf chlorophyll content, but the individual plant nodule number, individual plant nodule weight, individual plant leaf number, plant height, root length, aerial dry weight, and root dry weight were increased highest when added 10 mg L^-1 and 1 mg·L^-1 gibberellin into 12531f and gn5f, respectively. When 10 mg·L^-1 gibberellin added into inoculant of 12531f, the individual plant nodule number was higher than that of control and single inoculated 75.71% and 11.82%, respectively, but there were no significant difference (P＞0.05). the individual plant nodule weight was significantly higher than control and single inoculated 1 136.11% and 55.05%, respectively (P＜0.05). the individual plant leaf number was significantly higher than control and single inoculated 113.94% and 78.28%, respectively (P＜0.05). the plant height was significantly higher than control and single inoculated 83.33% and 50.24%, respectively (P＜0.05). the root length was significantly higher than control and single inoculated 115.28% and 29.17%, respectively (P＜0.05). the aerial dry weight was significantly higher than control and single inoculated 214.27% and 206.43%, respectively (P＜0.05). the root dry weight was significantly higher than control and single inoculated 1 156.19% and  1 049.53%, respectively (P＜0.05). When 1 mg·L^-1 gibberellin added into inoculant of gn5f, the individual plant nodule number was higher than control and single inoculated 82.86% and 4.07%, respectively, but there were no significant difference (P＞0.05). the individual plant nodule weight was significantly higher than control and single inoculated 697.22% and 105.00%, respectively (P＜0.05). the individual plant leaf number was higher than control and single inoculated 32.12% and 19.13%, respectively. the plant height was higher than control and single inoculated 95.24% and 37.82%, respectively; the root length was higher than control and single inoculated 76.39% and 5.83%, respectively, but there were no significant difference of these data (P＞0.05). the aerial dry weight was significantly higher than control and single inoculated 125.98% and 121.80%, respectively (P＜0.05). the root dry weight was significantly higher than control and single inoculated 864.43% and 762.21%, respectively (P＜0.05).【Conclusion】These results suggest that 10 mg·L^-1 gibberellin and 1 mg·L^-1gibberellin promote the migration and colonization of 12531f and gn5f in alfalfa seedling, also enhance the number of nodules and leaves, and promote the growth of plant height, root length and biomass, indicating that suitable gibberellin might be exploited to promote the migration and colonization of rhizobia in alfalfa tissue and positively impact growth and yield.

Engineering Design Parameters of Farming Plots Construction and Plot Characteristics in Hilly Area: A Case Study of Chongqing

ZHONG ShouQin, LIU Juan, LIU WeiPing, WEI ChaoFu

Scientia Agricultura Sinica. 2017, 50(23):  4558-4574.  doi:10.3864/j.issn.0578-1752.2017.23.009

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【Objective】In hilly area, the parameters designing of farming plots construction engineering plays a decisive role in the mechanization, large scale production and industrialization of agriculture. To date, with regard to most studies on the plot characteristics and soil properties, the basic engineering design parameters are often ignored. Thus, it is very necessary to find out technical points of the engineering design parameters of farming plots construction, which can promote sustainable development of cultivated land resources in hilly area.【Method】In this paper, we took 28 farming plots constructed between 2010 and 2015 in hilly areas of Chongqing as the research object, and the engineering design parameters and its effects on the plot characteristics were studied based on the investigation. 【Result】(1) Location selection method: The region where the terrain slope is more than 25° should be regarded as the forbidden construction zone. The region where the terrain slope is less than 25° should be regarded as the conditional construction zone, and the region of 15° is the key construction zone. Strip lands and gentle-slope lands were laid in the areas (called Area A), where the terrain slope was less than 6° and the concentrated area of cultivated land more than 3.33 hm² (50 mu). On the basis of the deduction Area A, the areas (called Area B) with terrain slope less than 15° and concentrated area of cultivated land more than 3.33 hm² (50 mu) were mainly suitable to lay strip lands, gentle-slope lands, level terraces and sloping terraces. On the basis of the deduction Area A and Area B, the areas (called Area C) with terrain slope less than 25° and concentrated area of cultivated land more than 3.33 hm² (50 mu) were mainly suitable to lay level terraces and sloping terraces. (2) Engineering design parameters: The length of strip lands, level terraces, sloping terraces and gentle-slope lands should be set to 50-200, 50-200, 30-200 and 50-300 m, respectively. The width should be set to 30-100, 10-30, 5-20 and 50-100 m, respectively. The slope of strip lands and level terraces should be set to 0°. But for sloping terraces and gentle-slope lands, the slope should be set to less than 10° and 6°, respectively. Ramming ridge was widely used in strip lands and gentle-slope lands construction. But if the soil properties can not meet the requirements of construction, rocks and squared stones can also be used for construction. Cemented squared stones and dry-laid rag were used in level terraces and sloping terraces, respectively. But if the squared stones can not be found around the farming plots, and the transport costs would be too much high, cemented rocks and ramming ridge can also be used for construction. (3) Effects on the plot characteristics: The farming plots construction engineering broke the original ridges of fields, and the scattered, small and irregular plots have been merged and re-planned. Finally, the larger, well-structured, relatively concentrated tillage plots can be formed. After the farming plots construction, the mean plot scale increased from 0.23 hm² to 0.63 hm², increased about 1.79 times. The shape index of the plot decreased from 18.02 to 10.22, decreased about 7.80. The density of the plot decreased from 7.09 plots/hm² to 3.35 plots/hm², decreased about 3.74 plots/hm². The Moran’s I index of plots’ area increased from 0.1937 to 0.3501, increased about 0.1564.【Conclusion】According to the terrain slope and the smallest construction unit of farming plots, four types of engineering (strip lands, level terraces, sloping terraces and gentle-slope lands) can be considered to be built in the hilly areas. They can significantly improve the fragmentation of cultivated land, and effectively promote the mechanization, large scale production and industrialization of agriculture. In addition, the engineering design should be based on the consideration of topography, soil properties, especially local materials for reducing construction costs in the hilly areas.

PLANT PROTECTION

Construction of Rice Leaf Sheath Protoplast Transformation System and Transient Expression of Pik-H4 and AvrPik-H4 Proteins

LIU Wei, LIU Hao, DONG ShuangYu, GU FengWei, CHEN ZhiQiang, WANG JiaFeng, WANG Hui

Scientia Agricultura Sinica. 2017, 50(23):  4575-4584.  doi:10.3864/j.issn.0578-1752.2017.23.010

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【Objective】 The objective of this study is to obtain the suitable digestion and transformation time of protoplasts of rice sheath, improve the efficiency of transient expression, the target gene can be detected at the protein level and expressed in large quantities. To explore the feasibility of transient expression of rice blast resistance protein Pik₁-H4, Pik₂-H4 and avirulence protein AvrPik-H4 in protoplasts of rice leaf sheath, and to analyze the function of above target genes.【Method】High blast resistance rice variety H4 and control variety Zhonger Ruanzhan were used as experimental materials. Rice seedlings were cultured with 1/2 MS medium at 25℃ for 7-10 d. The protoplasts were isolated by cellulase and macerozyme enzymatic action. The optimal time of digestion was obtained by counting the number of cells in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 h using hemocytometer. The target genes Pik₁-H4, Pik₂-H4 and AvrPik-H4 were fused with GFP to construct the transient expression vector. The total RNA was extracted from transformed protoplasts in 10, 12, 14, 16, 18, 20, 22 and 24 h, respectively. Real-time quantitative PCR (qRT-PCR) was used to detect the relative expression of GFP to obtain the best transformation time, UBQ housekeeping gene was used as control and GFP specific amplification primers were designed. The method of subcellular localization of the target gene by laser confocal scanning microscopy was used to estimate the gene function. The total protein was extracted and anti-GFP was used as the primary antibody, verified that the target protein successful expression by Western blot. 【Result】 rice seedlings were grown better quality and vitality in constant temperature 1/2 MS medium with rich and balanced nutrients, compared with soil planted at room temperature. Digested time had a greater impact on protoplast isolation efficiency. The results showed that the best time to digest was 4-6 h. The number of cells grew fastest at 3-4 h, tended to be stable at 4-6 h, showed a downward trend after 6 h, cell death rate accelerated and observed debris increased in the microscopic cell after 7 h. By detecting the relative expression of GFP, it was found that the most suitable time for transformation was 14-16 h, reached the highest value at 16 h, and then gradually decreased. Subsequently, fluorescence of the GFP protein was observed to be quenched by fluorescence microscopy. Subcellular localization observation of AvrPik-H4 protein was mainly located in the cell membrane, presumably this is a membrane protein that is transported by some form to the host cell as an exciton to trigger a series of reactions. Pik-H4 is composed of Pik₁-H4 and Pik₂-H4, which is highly efficient broad-spectrum rice blast gene. Pik₁-H4 and Pik₂-H4 were mainly located in the endoplasmic reticulum and plastid, respectively. From the subcellular localization results, it was presumed that Pik₁-H4 might be mainly involved in the recognition of Avr-Pik protein and signal transmission, Pik₂-H4 mainly play a role in changing the energy transmission and regulation of downstream disease caused hypersensitive reaction. Western blot results showed that the target protein was successfully expressed and the molecular size was correct. The expression of Pik₁-H4 and AvrPik-H4 was higher than that of Pik₂-H4, indicating that the size of the molecular weight is not a key factor affecting the transforming efficiency. 【Conclusion】 The protoplast transient expression system of rice leaf sheath has the characteristics of high efficiency and rapidity, the exploration of protoplast isolation and transformation time has laid a foundation for the extensive practice of rice transient expression system. The successful expression of the target gene has provided a valuable theoretical basis for the study of the interaction mechanism between Pik-H4 and Avr protein.

Comparison of Two Methods in Analysis of Binding Characteristics of Odorant Binding Proteins AlucOBP21 of Apolygus lucorum

LIU HangWei, ZHANG Qiang, Geng Ting, DONG Kun, AN XingKui, WANG Qi, ZHANG YongJun, GUO YuYuan

Scientia Agricultura Sinica. 2017, 50(23):  4585-4592.  doi:10.3864/j.issn.0578-1752.2017.23.011

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【Objective】The objective of this study is to compare two methods of fluorescence competitive binding assay and microscale thermophoresis (MST) in analysis of binding characteristics of odorant binding proteins Alucobp21 of Apolygus lucorum, and to explore a new method for determination of binding function of insect obps. 【Method】 Total RNA was extracted from antennae of both female and male A. lucorum adults by using Trizol reagent. The cDNAs were synthesized using the Superscript III Reverse Transcriptase system. AlucOBP21 was PCR-amplified using gene-specific primers. The sample cDNA of the antennae was used as the template. The PCR product was cloned into an expression vector PET-32a (+) for expression in prokaryotic BL21 (DE3) cells. The transformation of the strain with pET32a/AlucOBP21 was incubated in cultures and the crude protein with His-tag was obtained. The supernatant was obtained by sonication, purified by Ni ion affinity chromatography. Soon after the His-tag was removed with recombinant enterokinase, then the purified AlucOBP21 without His-tag was harvested. To investigate the binding abilities of AlucOBP21 to candidate ligand chemicals, fluorescence binding test was performed using 1-N-phenylnaphthylamine (1-NPN) as a fluorescence probe. 1-NPN and odor standard samples were dissolved in methanol (mass spectrometry grade). And also, the binding characteristics of recombinant AlucOBP21 to candidate ligands were explored by MST technique. In this assay, the candidate ligand samples were dissolved in dimethyl sulfoxide (DMSO) solution. Candidate ligands included 8 potential sex pheromones and sex pheromone analogues of mirids, 12 green leaf volatiles and a repellent (dimethyl disulfide) of A. lucorum.【Result】Recombinant AlucOBP21 was expressed in both supernatant and inclusion bodies. Finally, the supernatant fraction was selected to purify the target protein. Recombinant AlucOBP21 without His-tag was obtained by using recombinant entherokinase at 22℃. In fluorescence competitive binding assays, recombinant AlucOBP21 could bind with 1-NPN probe, dissociation constant was (6.88±0.31) μmol·L^-1. AlucOBP21 could bind with b-ionone and b-caryophyllene, and the dissociation constants were (13.74±1.93) and (13.24±2.12) μmol·L^-1, respectively. However, remaining candidate ligands could not effectively bind to recombinant AlucOBP21. Analysis of MST demonstrated that AlucOBP21 could bind with b-ionone, b-caryophyllene, b-pinene and limonene, and the dissociation constants were (0.20±0.02), (0.05±0.01), (0.70±0.04) and (0.40±0.06) μmol·L^-1, respectively. The thermophoresis of remaining candidate ligands with AlucOBP21 could not display regular change along with the change of ligand concentration. Therefore, these odorant chemicals could not bind with recombinant AlucOBP21.【Conclusion】In comparison with fluorescence competitive binding assay, MST detection demonstrated a broader ligand spectrum which could not miss odorant ligands with weak binding affinities.

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

The Changes of Organic Carbon and Its Fractions in Yellow Paddy Soils Under Long-Term Fertilization

WANG XiaoLi, GUO Zhen, DUAN JianJun, ZHOU ZhiGang, LIU YanLing, ZHANG YaRong

Scientia Agricultura Sinica. 2017, 50(23):  4593-4601.  doi:10.3864/j.issn.0578-1752.2017.23.012

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【Objective】The organic carbon of different protection mechanisms showed different fertility and bioavailability due to the difference in stability. The study of the response of soil organic carbon and its fractions in yellow paddy soils (main type of paddy soils in Guizhou province) on long-term fertilization and its evolution characteristics will provide scientific basis for the assessment of carbon sequestration potential and the improvement of soil fertility in paddy field in southwest China. 【Method】Soil samples of five fertilization treatments, including no fertilizer control (CK), chemical fertilizer (NPK), organic manure (M), low application rate of manure combined with chemical fertilizer (0.5MNPK) , and normal application rate of manure combined with chemical fertilizer (MNPK) , were collected every two years from 2006 to 2014 in the long-term fertilization experiment (starting from 1995) of yellow paddy soil in Guizhou Province, China. The content of soil total organic carbon and its four fractions organic carbon (unprotected organic carbon, physically-protected organic carbon, chemically-protected organic carbon and biochemically- protected organic carbon) were determined by Stewart physical-chemical fractionation method and elemental analyzing method. The linear regression equations were used to model the evolution characteristics of soil total organic carbon and organic carbon fractions. 【Result】The results showed that compared with CK and NPK treatments, soil total organic carbon contents under the manuring treatments (0.5MNPK/M/MNPK) were significantly increased by 15%-39%, among which MNPK treatment had the maximum increasing range, and the unprotected organic carbon, physically-protected organic carbon and chemically-protected organic carbon were significantly increased under the manuring treatments (0.5MNPK/M/MNPK). The soil organic carbon and its fractions contents in four fertilization treatments (NPK/0.5MNPK/M/MNPK) increased with time from 2006 to 2014, among which the trends of linear increase of unprotected organic carbon, physically-protected organic carbon and chemically-protected organic carbon under the manuring treatments (0.5MNPK/M/MNPK) were more obvious than NPK treatment. However, which of CK treatment had no evident change with fertilization time. The average annual growth rate and growth range of soil unprotected organic carbon, physically-protected organic carbon and chemically-protected organic carbon under manuring treatments (0.5MNPK/M/MNPK) were both higher than those of CK and NPK treatments. The average annual increasing rates of soil total organic carbon under manuring treatments (0.5MNPK/M/MNPK) were 3.5-3.7 times and 1.5-1.6 times than those of CK and NPK treatments, respectively. The ratio of unprotected organic carbon to total organic carbon (58%-63%) was 3-14 times than those of other organic carbon fractions. 【Conclusion】The unprotected organic carbon not only accounted for the largest portion in soil organic carbon, but also had the most highly sensitivity to fertilization. The normal application rate of manure combined with chemical fertilizer is an effective model for improving soil organic carbon storage.

Effects of Straw Mulching and Vetiver Grass Hedgerows on Soil Enzyme Activities and Soil Microbial Community Structure in Red Soil Sloping Land

CHENG YanHong, HUANG QianRu, WU Lin, HUANG ShangShu, ZHONG YiJun, SUN YongMing, ZHANG Kun, ZHANG XinLiang

Scientia Agricultura Sinica. 2017, 50(23):  4602-4612.  doi:10.3864/j.issn.0578-1752.2017.23.013

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【Objective】 The objective of this paper is to clarify the relationship between soil organic carbon and microorganism under different biological water protection measures. 【Method】 Soil samples were collected from a field soil and water conservation measures experiment, which was started since 2009 with 4 treatments, including peanuts grown in high order (CK), hedgerows+ peanuts (H), mulching+ peanuts (M) and hedgerows+ mulching+ peanuts (HM). Phospholipid fatty acid (PLFA) profiles for each treatment were used to characterize the microbial community structure. Soil organic carbon, microbial biomass carbon, dissolved organic carbon and soil enzyme activities were also determined. 【Result】 Compared with the CK, all treatments showed total organic carbon, dissolved organic carbon, total microbial biomass carbon and qMB increased. H is the main influence factor on MBC and Qmb. The results showed that H significantly affected the activities of soil invertase, cellulase, β-Glu and intensity of MBC/TOC. Compared with the CK, H, M and HM, PLFA increased the total PLFAs, bacterial and actinomycetes PLFAs, while decreased G- PLFA/G+PLFA, and that was the lowest under HM treatment. Principal components analysis showed that soil microbial community structure was similar to that of H and M, which was larger difference with CK. Redundancy analysis showed that TOC, MBC and enzymes activities of soil CEL, INV, β-Glu were all the main factors affecting the quantity and structure of soil microorganisms. 【Conclusion】 Microbial biomass, community structure and soil normalized C-acquiring enzyme activity were influenced significantly by straw mulching and vetiver grass hedgerows.

HORTICULTURE

Gene Mapping of Aphid-Resistant for Peach Using SNP Markers

ZHANG NanNan, LU ZhenHua, CUI GuoChao, PAN Lei, ZENG WenFang, NIU Liang, WANG ZhiQiang

Scientia Agricultura Sinica. 2017, 50(23):  4613-4621.  doi:10.3864/j.issn.0578-1752.2017.23.014

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【Objective】 The objective of this study is to identify the SNP loci tightly linked to peach( Prunus persica (L.) Batsch ) aphid(Myzus persicae (Sulzer)) resistance traits, revealing its genetic basis and laying a foundation for the marker assistant selection in resistance breeding of peach.【Method】In this study, the population used for the mapping study consisted of141 individuals which were obtained from a cross between female parent (‘01-77-3’ ) and male parent (‘CN13’). Referencing the peach genome and using Sanger sequencing, single nucleotide polymorphism (SNP) markers were developed in female and male parents and 8 progenies to obtain markers linked to the target loci which were tested on the whole population. Subsequently, using whole genome re-sequencing data of two parents, SNPs for fine mapping were selected based on the genotype of two parents, andemployed to conduct genotyping to obtain the SNP marker linked to resistance traits. Ultimately, the fine mapping region was validated by using an InDel marker to verify the genotype of F₁ population generated from ‘96-5-1’ × ‘10-7’. 【Result】 As a result of phenotype identification of 141 progenies, the segregation ratio of resistance to aphid to susceptible ones showed 1﹕1 (P: 0.556; χ²: 0.348). Using Sanger sequencing we mapped the resistant gene to an approximate 9.92 Mb physical distance between two SNP markers, Pp01_38011783 and Pp01_47231340 on Pp01. For fine mapping, a total of 17.109 Gb clean data was generated from genome re-sequencing and the average coverage depth is 75.19×. 11 of 29 pairs of primers which were designed based on genome re-sequencing data were effective and linked to target trait. As a result of genotyping, we obtained two SNP makers tightly linked to desired trait, SNP_Pp01_45665389 and SNP_Pp01_46120950, with genetic distance of 1.4 cM and 2.1 cM, respectively. The target locus was between these two markers, an approximate physical distance of 460 Kb, and the gene was co-segregating with another marker SNP_Pp01_45712702. With fine gene mapping region, an InDel marker, KYYZ_Pp01_45799758, was designed and used to verify the phenotype of 92 individuals generated from an F₁ segregation population of ‘96-5-1’ ב10-7’ with 98.91% accuracy.【Conclusion】The SNP loci and candidate genes related closely with aphid-resistant gene of peach were identified in this study. The resistant gene had been mapped to an approximate 460 kb physical distance between two SNP markers, SNP_Pp01_45665389 and SNP_Pp01_46120950 at the bottom of Pp01 which contains 56 transcripts (52 candidate genes).

The Physical and Chemical Properties of Different Substrates and Their Effects on Agronomic Traits and Yield of Agaricus bisporus

ZHANG HaoLin, CHEN QingJun, ZHANG GuoQing, QIN Yong, GAO XiaoJing, QIN GaiJuan, WU XinRui

Scientia Agricultura Sinica. 2017, 50(23):  4622-4631.  doi:10.3864/j.issn.0578-1752.2017.23.015

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【Objective】In order to provide theoretical basis and technical support for Agaricus bisporus cultivation in formula optimization and resource utilization.【Method】Four formulas of different substrates were performed as substrate materials, including wheat straw formula (T1), wheat and rice straw mixing formula (T2), wheat straw and corn stalk mixing formula (T3), and wheat straw and spent compost mixing formula (T4). The substrates were composted using the secondary fermentation method in the factory fermentation tunnel. Mushroom management proceeded in standard mushroom room workshop with controllable temperature, humidity and ventilation. The culture strain was Sylven A15. Substrate samples were collected at different time during composting and mushroom cultivation. Seven physical and chemical properties were measured, including water content, pH value, conductivity, carbon content, ash content, nitrogen content, and C/N ratio. The relationship between the physicochemical properties and corresponding yields were analyzed by multiple regression analysis. The agronomic traits of mushroom, such as mushroom weight, pileus diameter, pileus thickness and fruit body hardness, were analyzed based on the UPOV method. 【Result】The results showed that both water content and pH of the substrates in the four formulas were declined from the composting to cultivation periods. At the end of secondary fermentation, water content of the four formulas was about 70%, and the pH of formula T1 was 9.02. They were significantly higher than other formulations. The conductivity started to rise at the end of secondary fermentation. The conductivity of the four formulas softly increased during the secondary fermentation except T4 which underwent a significant declining at first fermentation stage. The ash content of the samples presented an upward trend. While at the end of the secondary fermentation, the ash content of formula T1 was significantly lower than that of the other three. The carbon content was continuously decreased during the culturing period especially in fruiting stage. At the end of the secondary fermentation, the carbon content of formula T1 was significantly higher than that of others. The nitrogen content of substrates at the end of the secondary fermentation was an important indicator for mushroom yield of the 1^st flush. The amount reached to 1.9%-2.2%. During the fruiting stage, the nitrogen content was gradually reduced due to the consumption of substrates nutrition by mushroom mycelia. The nitrogen content of formula T4 was significantly higher than that of others. The formula T1 possessed the highest water content at cultivation period, and the highest yield of mushroom with the most stable agronomic characters. The second flush yield of formulas T1, T2 and T3 were 3 061.41, 2 534.47, 2 534.47 kg, respectively. They accounted for 43.81%, 39.89% and 49.71% of their total yield, respectively. The first flush yield of formula T4 was the the highest (3 064.19 kg), and accounted for 47.39% of its total yield. The multiple regression analysis resulted Y₁=-5926.766＋3770.091X₆, Y₂=6285.502＋4920.672X₁-1061.418X₂-245.782X₃＋949.998X₅＋26081.326X₆, Y₃=3073.013＋7030.476X₁-114.728X₅-910.576X₆. The results showed that the water content of substrates was positively correlated with the yield of the 1^st, 2^nd and 3^rd flush. The nitrogen content of substrates was positively correlated with the yield of the 1^st and 2^nd flush. The carbon content of substrates was positively correlated with the yield of the 2^nd flush, while the carbon and nitrogen content of substrates was negatively correlated with the yield of 3^rd flush. 【Conclusion】The water content of substrates during fruiting stage is the key element to improve the agronomic traits and yield ofmushroom A. bisporus. Increasing the content of carbon and nitrogen is beneficial for the yield formation of the 1^st and 2^nd flush.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Regulation of the Promoter of FAM213B Gene in Porcine Endometrial Cells

ZHANG AiLing, SUN XianYue, LU XiaoZhang, WU Qi, LI JiaQi, ZHANG Hao

Scientia Agricultura Sinica. 2017, 50(23):  4632-4643.  doi:10.3864/j.issn.0578-1752.2017.23.016

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【Objective】To interpret partially the role of FAM213B gene expression in prostaglandin synthetise and sow pregnancy through the identification of the transcription region of porcine FAM213B gene promoter and the effection of NFκB on the promoter. 【Method】 The porcine endometrium from the uterus in follicular phase was digested by collagenase and the isolated endometrial cells were cultured for the detection of the FAM213B promoter activity. Based on the mRNA and promoter sequences of FAM213B gene obtained in our former work (GenBank ID: KX444503 and 100134955), the longer 5′ regulationary sequence was amplified and sequenced. Then, seven promoter fragments with 5′ terminal deletion, containing Mlu I and Xho I sites, were linked into the Dual-luciferase Reporter vectors. Seven constructed vectors treated by endotoxin free and pRL-TK plasmid were co-transfected into endometrial cells through liposome method. The core region of transcriptional activity of the gene promoter was identified through the Dual-luciferase Reporter Assay System. The putative transcription factors binding sites were analyzed by bioinformatics, and the binding of NFκB with FAM213B promoter were detected by ChIP (Chromatin immunoprecipitation). The over-expression vectors of NFκB1and RelA and interference fragments of their own were transfected into endometrial cells. Then, the transcription activity of the promoter and mRNA expression of the gene were detected by the Dual-luciferase Reporter system and fluorescent quantitative, respectively. 【Result】 Through the PCR and sequencing, one fragment of 2 261 bp (-2178/+83) of porcine FAM213Bgene were obtained. The bioinformatics analysis showed that there were putative binding sites of CREB, CCAAT, E-box, and NFκB in the promoter. And the putative binding sites of NFκB were found in -1143/-1132 and -664/-655 regions. The result of the Dual-luciferase Reporter showed the region of P2 (-1352/+30) exhibited the strongest transcriptional activity, and it was significantly higher than that of P1 (-1760/+30) (P＜0.01), which showed there were negative regulation elements in -1760/-1352 region. And the transcriptional activity of P2 (-1352/+30) was significantly higher than that of P3(-919/+30) (P＜0.05), implying the existence of positive elements in -1352/- 919 region. The significant stronger activity of P3 (-919/+30) than P4(-604/+80) (P＜0.01) meant the existence of positive elements in -919/-604 region. No significant differences were observed between P4, P5, P6, and P7. The region of -1352/-919 was the core element of the promoter. The results of Chromatin Immunoprecipitation (ChIP) demonstrated that NFκB1 binds to one site around -1143/-1132, and RelA site around -664/-655. The co-transfection of the over expression of pcDNA3.1-NFκB1 and P2 vector into endometrial cells increased the activity of the promoter (P＜0.01), and the transfection of pcDNA3.1-NFκB1 enhanced the mRNA expression of FAM213B (P＜0.05). While the co-transfection of the over expression of pcDNA3.1-RelA and P3 vector into endometrial cells decreased the activity of the promoter (P＜0.05), and the transfection of pcDNA3.1-RelA weakened the mRNA expression of the gene. At the same time, the transfection of inference siRNA fragments of NFκB1 and RelA into endometrial cells led the contrary results for the activity of the promoter and the mRNA expression of the gene.【Conclusion】The core region of porcine FAM213B gene promoter was identified round -1352/-919. NFκB was the trancription factor of FAM213B gene. NFκB1 and RelA, two members of NFκB family, regulate the expression of FAM213B gene in endometrial cells.

The Comparative Transcriptome Analysis of Parabronema skrjabini at Different Developmental Stages

WANG WenLong, FENG ChenChen, HONG Mei, YUE JianWei, Huhebateer, LIU ChunXia

Scientia Agricultura Sinica. 2017, 50(23):  4644-4655.  doi:10.3864/j.issn.0578-1752.2017.23.017

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【Objective】 The objective of this study was to identify the differentially expressed genes(DEG) and describe biological characteristics involved in functional classifications and metabolic pathways at different developmental stages of Parabronema skrjabini infecting camel, which is necessary to better understand functional genes involved in growth and development and enrich the transcriptome data of parasitical nematodes.【Method】Eggs, the third-stage larvae(L3s) and females of Parabronema skrjabini were sequenced by Illumina HiSeq2000^TM sequencing platform and constructed their cDNA libraries after quality filtering. De novo assembling and assembly efficiency assessment were carried out using Trinity, a short-read assembly program. Then all the effective sequential data obtained were assigned to the relevant databases to perform functional annotation and bioinformatic analysis.【Result】The results showed that 47 717, 76 342 and 54 624 unigenes were obtained respectively in eggs，the third-stage larvae and female stages. 33 579 differentially expressed genes(DEGs) were identified by comparing the unigenes obtained from eggs and L3s stages, of which 20 477 were up-regulated and 13 102 were down-regulated. There were 32 199 differentially expressed genes between L3s and female stages, of these genes, 9 293 were up-regulated genes and 22 906 were down-regulated genes. The differentially expressed genes of two pairwise comparisons were respectively enriched in Gene Ontology. 6 617, 3 891 and 8 755 differentially expressed genes comparing eggs and L3s stages were annotated into database of biological process, cellular component and molecular function respectively, while the number by comparing L3s and female stages were 7 043, 3 686 and 10 177 respectively. In KEGG pathways identification, 6 521 differentially expressed genes comparing eggs and L3s stages were assigned to 251 KEGG pathways, and clustered significantly in MARK, Wnt signaling pathways and oxidative phosphorylation. In comparison of L3s and female stages, 6 528 differentially expressed genes were enriched in metabolic pathways, DNA replication and cell cycle. Functional cluster analysis indicated that the regulation related differential genes of growth rate and the reproductive and genital development were highly expressed in eggs and females stages, then the genes of the defense and carbohydrate metabolism were enriched in exclusive L3s stage. Differential genes of embryonic and post-embryonic development were highly expressed in all three stages, and 196 function genes from embryonic development and 166 function genes from post-embryonic development were co-expressed in all three stages, implied that these genes played a crucial role in (post-)embryonic development. Moreover, we identified 9 heterochronic genes, such as LIN-28, LIN-14 and RHEB-1, 48 nuclear hormone receptors (NHRs) genes, including NHR-49, NHR-48, NHR-40 and NHR-1, and 36 zinc metalloproteinase(NAS) genes, including NAS-36, NAS-33 and NAS-14. Then the analysis of enrichment capacity in three stages showed these genes were necessary to regulate the different development stages of Parabronema skrjabini. 【Conclusion】The transcriptomic research of Parabronema skrjabini at three developmental stages using RNA-seq revealed biological characteristics of differentially expressed genes involved in development- related GO functional classification, KEGG pathway and functional cluster and identified many kinds of heterochronic genes and developmental genes, which provides a foundation and reference for further investigation of the whole genome sequence analysis, interaction between Parabronema skrjabini and host, pathogenic mechanism and immune evasion.

Comparative Analysis of Phosphoproteome Between Mandibular Glands of High Royal Jelly Producing Bees and Italian Bees

LI Shuang, LI JianKe

Scientia Agricultura Sinica. 2017, 50(23):  4656-4670.  doi:10.3864/j.issn.0578-1752.2017.23.018

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【Objective】The principal biological function of worker bees’ mandibular glands is to secrete fatty acids to provide the nutrition for the colony and participate in synthesis of alarm pheromone. High royal jelly producing bees (RJBs, Apis mellifera liguatica) and Italian bees (ITBs, Apis mellifera liguatica) are both major honeybee species in China, but the mechanism of regulating the development and function of mandibular glands by phosphoproteome is not reported yet. The objective of this study is to compare the differences of mandibular glands of phosphoproteome, reveal the mechanism of the regulation of mandibular glands by phosphoproteome and fatty acids metabolism.【Method】The mandibular glands of newly emerged, nurse, and foragers of RJBs and ITBs were collected, then after, mandibular gland proteins were extracted and digested by an enzyme. Phosphopeptides were enriched by IMAC (immobilized metal-affinity chromatography) and desalted by using Zip-tip C18 columns. Peptides from each of the samples were analyzed by LC-MS/MS (liquid chromatography-mass/mass). Furthermore, MaxQuant and Perseus bioinformatics tools were used to evaluate phosphoproteome quality. These bioinformatics tools were further utilized for principal component and profiling hierarchical clustering quantification analyses. In addition, PEAKS software was employed for protein quantification and quality analyses. Finally, biological processes and KEGG metabolic pathways in mandibular glands of RJB and ITB were compared. Phosphosites were determined and motifs were predicted by using Scaffold PTM bioinformatics tool.【Result】 The numbers of identified phosphoproteins of RJBs (2 225, 1 922, 2 159) were significantly higher than those of 1 740, 1 592, 1 682 in ITBs, illustrating that RJBs’ mandibular gland phosphorylation regulation network was likely more complicated than ITBs’. Although the phosphorylation process in mandibular glands of RJBs was significantly different from that in ITBs, a similar phosphoproteomes profiling across the gland development suggested that the phosphoproteins at distinct stages in RJBs and ITBs performed similar biological function to ensure the gland development and secretion activity. the glands of each stage in RJBs and ITBs had significant phosphoproteome differences, of which nurse bees were the most significantly diverged. Eighty-seven phosphoproteins were highly abundant in RJBs, and most of them were mainly related to energy and fatty acid metabolic process. These indicated the key roles of fatty acid metabolism in boosting the ability of fatty acid synthesis (including 10-HDA) in mandibular glands to prime the quantity of 10-HDA in royal jelly alongside the increased royal jelly production, which was essential for providing qualitative nutrition for the survival of population. Forty-one highly abundant phosphoproteins in ITBs were mainly related to energy metabolism. Three kinds of motifs were detected at each stage of glands in both RJBs and ITBs: acidic, basic and pro-directed. The unique kinase family recognized by motifs in nurse bees of RJBs was associated with cell proliferation, which might support gland morphological development and related to increased secretion capacity of RJBs.【Conclusion】RJBs and ITBs have shaped different phosphoproteome signatures to maintain mandibular gland development and function at each stage. The most profound divergence occurs during the nurse stage, which RJBs may increase the ability of fatty acid synthesis to ensure the basic function of royal jelly. The data help us gain a novel understanding of the molecular underpinnings to drive enhanced high royal jelly production in RJBs at the level of protein modification.