Table of Content

01 September 2023, Volume 56 Issue 17

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SPECIAL FOCUS: HERBICIDE-TOLERANCE COTTON CREATION BY GENETIC TRANSFORMATION AND GENOME EDITING

Advancements in Herbicide-Tolerant Cotton Research and Breeding in China

LIANG ChengZhen, JIN ShuangXia

Scientia Agricultura Sinica. 2023, 56(17):  3247-3250.  doi:10.3864/j.issn.0578-1752.2023.17.001

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Identification of Target Traits and Genetic Stability of Transgenic Cotton GGK2

LIANG ChengZhen, ZANG YouYi, MENG ZhiGang, WANG Yuan, MUBASHIR Abbas, HE HaiYan, ZHOU Qi, WEI YunXiao, ZHANG Rui, GUO SanDui

Scientia Agricultura Sinica. 2023, 56(17):  3251-3260.  doi:10.3864/j.issn.0578-1752.2023.17.002

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【Objective】The objective of this study is to confirm the target traits and genetic stability of transgenic glyphosate- resistant cotton GGK2 and provide technical support for its commercialization. 【Method】T₃, T₄, and T₅ transgenic cotton plants GGK2 were subjected to insertion site-specific PCR, Southern blot, ELISA, bioassays in the laboratory and field, analysis of target herbicide tolerance, and investigation of nutritional constituents. 【Result】The results indicated that the target genes, GR79 EPSPS and GAT, were integrated into the cotton genome as single copies and stably inherited in GGK2 plants. In GGK2 cotton, GR79 EPSPS, GAT, and NPTⅡ proteins were expressed at different stages and in different tissues, with relatively high expression levels in the leaves. At the four-leaf stage, bud stage and boll opening stag, the expression levels in leaves were 128.7-192.4 µg·g^-1, 24.4-35.0 µg·g^-1, and 17.0-23.9 µg·g^-1 fresh weight for GR79 EPSPS, GAT, and NPTⅡ, respectively. In the field, transgenic cotton GGK2 tolerated up to four times the recommended medium dose of glyphosate application. No significant differences were observed in agronomic traits and nutritional constituents compared to the control, Coker312. 【Conclusion】These data demonstrate that transgenic cotton GGK2 is genetically stable and highly resistant to herbicides. Therefore, it can be utilized for breeding high-glyphosate- resistant commercial cotton varieties.

The Creation and Characteristics of Cotton Germplasm Lines Transgenic 1174AALdico-2+CTP Gene with Excellent Glyphosate Tolerance

WANG WanRu, CAO YueFen, SHENG Kuang, CHEN JinHong, ZHAO TianLun, ZHU ShuiJin

Scientia Agricultura Sinica. 2023, 56(17):  3261-3276.  doi:10.3864/j.issn.0578-1752.2023.17.003

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【Objective】 Weeds are one of the factors limiting cotton growth in cotton production, which not only competes with cotton for nutrition, water, and light, affecting the growth and development of cotton but also the yield and quality of cotton. The aim of this study is to develop excellent cotton germplasms with high glyphosate tolerance by genetic engineering and provide technical support for the commercialization of glyphosate-tolerant cotton to realize chemical weed control and enhance the economic benefits of cotton production. 【Method】 To construct the cotton transformation vector, the EPSPS gene 1174AALdico-2 from Deinococcus radiodurans was connected to the chloroplast transit peptide (CTP), 35S was used as the promoter, and the two target genes were linked in series. The target gene was transformed into the CCRI 49 by Agrobacterium tumefaciens in vivo transformation, and the positive transformants were obtained. The positive transgenic plants obtained were evaluated for glyphosate tolerance, molecular characterization, agronomic and economic traits, in order to obtain outstanding transgenic cotton germplasms with excellent glyphosate tolerance, using the recipient cultivar, CCRI 49, and the non-transgenic NON isolated from the transformants during selfing as the controls. 【Result】 138 positive transformants were obtained by Agrobacterium tumefaciens in vivo transformation. All the transformants were tested for target gene PCR, Southern blot, Western blot, and ELISA detection etc., and 17 positive transformants with clear molecular characteristics and high expression of foreign genes were identified. Southern blot and nucleotide sequencing results revealed that there was great variation in insertion site and copy numbers among the 17 transformants. Among them, ZD131, ZD185, and ZD207 had single-copy insertion sites, and the foreign genes were located on D7, D13, and A12, respectively. The results of glyphosate tolerance identification revealed that the glyphosate tolerance of the three transformants, ZD131, ZD185, and ZD207, was stably inherited across three generations, and the glyphosate tolerance of the transformants was strong, with the ability to tolerate four times the recommended dose of glyphosate in the field. The agronomic and economic features assessment findings revealed that the ZD131, ZD185, and ZD207 grew properly and had excellent agronomic traits such as large boll, high lint percentage, and high boll setting, as well as their lint yield was higher than that of the receptor cultivar control and their fiber quality reached to the high-quality cotton level. 【Conclusion】 The EPSPS gene 1174AALdico-2 from Deinococcus radiodurans connected with the chloroplast transit peptide, and the two gene tandem together was transferred into CCRI 49 by Agrobacterium tumefaciens in vivo transformation technology. After a series of screens, three outstanding transgenic glyphosate-tolerant germplasms, ZD131, ZD185, and ZD207, were obtained. This method not only improves cotton's glyphosate tolerance but also improves the agronomic and economic aspects of transgenic materials.

Identification of Molecular Characterizations for Transgenic Cotton R1-3 Line of Glyphosate Tolerance

MA YanBin, LI HuanLi, WEN Jin, ZHOU XianTing, QIN Xin, WANG Xia, WANG XinSheng, LI YanE

Scientia Agricultura Sinica. 2023, 56(17):  3277-3284.  doi:10.3864/j.issn.0578-1752.2023.17.004

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【Objective】 To obtain the transgenic cotton by Agrobacterium-mediated method was the purpose using the new gene G10aroA with high glyphosate tolerance in our laboratory. Meanwhile, it was necessary to provide molecular characteristics of genomic integration of the exogenous gene for breeding utilization in future. 【Method】 The Agrobacterium-mediated method was used for transgenic cotton plants obtained via tissue culture with glyphosate herbicide. Western blot was utilized to detect the expression of exogenous proteins in different organs of transgenic cotton R1-3. The number of loci in cotton genomes were evaluated by Southern blot for detecting integration of the exogenous sequence from the pCAMBIA1300 construct. The flanking sequence near the insertion site was amplified by TAIL-PCR, which the extractions of DNA were cloned and sequenced. The location of the chromosome for the flanking sequences were compared and analyzed on the website of NCBI blast. 【Result】 Regenerated R1-3 cotton plants were successfully obtained by tissue culture depending on glyphosate screening. the specific protein coded by exogenous G10aroA gene could be detected normally via Western blotting in the leaves, bracts, flowers and stems separately, and the size of the exogenous protein around 46 kDa were also observed in this experiment. In addition, the result of Southern-blot confirmed that the exogenous fragment containing G10aroA sequences was single integration in the genome of transgenic cotton R1-3, in which the bands digested severally by KpnⅠ and EcoRⅠ endonucleases were distinctly observed near the position of 6 557 bp and 4 316 bp strips on the nylon membrane respectively. The analysis of flanking sequence alignment for the integration site was predicted to be located on the 11th chromosome of either cotton A or D genome, and the left and right boundaries of the insertion site were further located between 47 525 303 and 47 525 449 of the chromosomes. In addition, the specific identification for the fusion site showed that the target band of approximately 300 bp for testing left border junction, and a specific target band can be amplified about 600 bp for testing the right border fusion site. 【Conclusion】 In this study, we obtained R1-3 transgenic cotton plants that also have exhibited stable genetic characteristics of glyphosate resistance during the process of self-crossing breeding. The protein coded by G10aroA gene was about the size of 46 kDa that could be detected in different tissues of transgenic cotton R1-3 plant. Furthermore, the exogenous fragment including G10aroA gene was identified with a single location by southern blot in the cotton genomes, and the integration site was located at the 11th chromosome. The results of comparative analysis were predicted that a nucleotide sequence about 146 bp length was deleted at the integration of the genome.

Opportunities and Challenges for Developing Herbicide-Resistance Crops in the Post-Genomic Era

WU YuanLong, HUI FengJiao, PAN ZhenYuan, YOU ChunYuan, LIN HaiRong, LI ZhiBo, JIN ShuangXia, NIE XinHui

Scientia Agricultura Sinica. 2023, 56(17):  3285-3301.  doi:10.3864/j.issn.0578-1752.2023.17.005

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Global agriculture is facing severe challenges, and breeding technology is the foundation and key to the development of the seed industry. Gene editing technology refers to the precise modification of target genes to achieve deletion, insertion, and replacement of specific target gene fragments. It can precisely modify target genes or introduce certain excellent genes into crops to produce crops with excellent agronomic traits, which has great potential in molecular design breeding and is of great significance to ensuring food security. Weed damage has a huge impact on the yield and quality of crops. To control weed damage efficiently, safely and sustainably has always been a hot research topic. Currently, more than 200 types of chemical herbicides have emerged in the global market. Using chemical methods to control weeds has become an important part of modern agriculture, and the cost of weed control has been significantly reduced by promoting herbicide-resistant crops. However, with the large-scale promotion of herbicide-resistant crops and the long-term use of single herbicides, environmental safety problems such as weed resistance and escape of resistant genes have gradually been discovered. Currently, the development of functional genomics, bioinformatics and genetic engineering technology (especially the widespread application of gene editing technology in plants) has created conditions for the creation of herbicide-resistant crops and new efficient weed control systems. In this article, the main target genes of herbicides that inhibit amino acid biosynthesis, lipid metabolism, carotenoid, plastoquinone and tocopherol biosynthesis pathways and their action mechanisms are introduced at first. Secondly, two methods for mining new herbicide resistance genes and herbicide systems are introduced, including the directed mutation method of herbicide resistance genes within crops based on CRISPR/Cas system and the resistance gene guidance method based on the co-evolution theory of natural product and organisms in nature. Moreover, the research progress of three breeding methods for herbicide resistant crops was reviewed, including conventional breeding, transgenic breeding and CRISPR/Cas genome editing based breeding. Among them, the research progress of CIRSPR/Cas system, base editing technology, and prime editing system in cultivating herbicide resistant crops were highlighted. The main challenge faced by chemical control of weeds and herbicide resistant crops is resistant weeds and environmental safety issues, and gene escape, respectively. At present, the rapid development of genome editing technology provides new solutions and new opportunities for the development of herbicide resistant crops in the post genome era. Finally, the prospects for the future of herbicide-resistant crops were provided.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION

Critical Lowest Temperature for the Safe Heading of Various Types of Late-Season Rice Cultivars and the Safe Dates for Their Full Heading in Different Double-Season Rice Cropping Regions of Hubei Province

LI Yi, LU JianXin, CAO Peng, ZHOU DengWen, LIU JiMin, TIAN PingPing, CAI MingLi, CAO CouGui, YANG TeWu

Scientia Agricultura Sinica. 2023, 56(17):  3302-3316.  doi:10.3864/j.issn.0578-1752.2023.17.006

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【Objective】 The objective of this study is to determine the critical lowest temperature for the safe heading of various types of late-season rice cultivars and the dates for their safe full heading in different double-season rice production regions of Hubei Province, and to provide guidelines for the rational layout and scientific arrangement of double-season rice production in different regions. 【Method】 Different types of late-season rice cultivars including japonica, indica and indica-japonica hybrid those are suitable for different cropping methods in the double-season rice cropping regions of Hubei Province were used as materials and a multi-site field experiment with various sowing dates was conducted at Chibi City, Xianning, Gongan County, Jingzhou, Hongshan District, Wuhan, Tuanfeng County, Huanggang, and Xiaochang County, Xiaogan, Hubei Province, respectively, in 2020 and 2021. Meanwhile, simulated low-temperature treatment was imposed to the potted plants during booting and heading in a pot experiment. The grain-setting rate and grain yield per plant or hill were investigated and the relationship between grain-setting rate and air temperature prior to full heading was analyzed. The critical lowest temperature for their safe heading of various types of cultivars was determined using the temperature under that the grain-setting rate was reduced to 82% of that under the optimum temperature. Furthermore, the daily risk maps for the full heading of different cultivar types in September were drawn based on the meteorological data in the past 30 years in various double-season rice cropping regions of Hubei Province. The latest safe dates for full heading of various types of cultivars in different double-season rice cropping regions were determined as the injury from low temperature to their heading can be avoided in more than 80% of years. 【Result】 With the delay of sowing date, the heading date of various cultivars was delayed. The grain yield per plant or hill decreased significantly under late sowing conditions. Grain-setting rate, a determinant factor that affects the yield of individual plant or hill, was quadratically related to the mean daily temperature in the 4 days prior to full heading. The critical lowest temperature for the safe heading in the 4 days prior to full heading was 19.6 ℃ for japonica cultivars, which was 0.4 ℃ lower than the conventional standard; and 22.4 ℃ for indica cultivars, which was 0.4 ℃ higher than the conventional standard; and 22.2 ℃ for indica-japonica hybrid cultivars, which was close to that of indica cultivars. The latest safe date for the full heading of late-season japonica, indica and indica-japonica hybrid cultivars was September 25-28, 16-19 and 17-20 in southeastern Hubei; September 23-27, 13-19 and 14-20 in eastern Hubei; September 25-27, 10-17 and 10-18 in Jianghan Plain; September 22-26, 7-15 and 7-17 in northern Hubei, respectively. 【Conclusion】 The safe date of full heading for late-season rice differed much among various cultivar types and in different double-season rice cropping regions of Hubei Province. It showed a trend that became earlier from south to north in Hubei Province, and was earlier for indica cultivars than for japonica cultivars. The latest safe date of full heading for indica-japonica hybrid cultivars was close to that for indica cultivars. It is of importance to select suitable cultivars and arrange reasonable sowing dates according to the local climatic conditions and cultivar characteristics for the safe production of late-season rice. Plantation using japonica cultivars is helpful to combat the damage from low temperature during heading and flowering in the late-season rice production.

Effects of Multiple Green Manure After Wheat Combined with Different Levels of Nitrogen Fertilization on Wheat Yield, Grain Quality, and Nitrogen Utilization

ZHANG WenXia, LI Pan, YIN Wen, CHEN GuiPing, FAN ZhiLong, HU FaLong, FAN Hong, HE Wei

Scientia Agricultura Sinica. 2023, 56(17):  3317-3330.  doi:10.3864/j.issn.0578-1752.2023.17.007

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【Objective】 Aiming at the problems of large nitrogen input, single fertilizer source, low nitrogen utilization rate, and poor quality of wheat in spring wheat cultivation in Hexi areas of Gansu Province, the objective of this study is to explore the effects of suitable green manure and reduced nitrogen fertilizer cultivation techniques on grain yield and quality, and nitrogen absorption and utilization of spring wheat, and to provide a theoretical basis for high yield, high quality, and green production of wheat in Hexi irrigation areas. 【Method】 A split plot experiment was conducted from 2019 to 2021 in the Hexi oasis irrigation areas of Gansu Province. Two cropping patterns of multiple green manure after wheat (W-G) and sole wheat (W) were set in the main plot. There were five N fertilizer levels in the sub-plot: 100% of conventional N fertilizer by the farmer (180 kg·hm^-2, N₄), 85% of conventional N fertilizer (N₃), 70% of conventional N fertilizer (N₂), 55% of conventional N fertilizer (N₁), and no N fertilizer (N₀). 【Result】 Multiple green manure after wheat combined with 85% N application (W-G-N₃) was effectively increased wheat grain yield and biomass. The grain yield of W-G-N₃ was increased by 16.7%-18.4% and 13.6%-34.4%, respectively, compared with the 85% N application (W-N₃) and conventional N application (W-N₄) treatments for the sole wheat. The biomass of W-G-N₃ was increased by 11.3% (2020) and 5.2%-11.6% (2020 to 2021), respectively, compared with the W-N₃ and W-N₄ treatments. The increase of grain yield was greater than that of biomass, thus, the W-G-N₃ treatment had higher harvest index, which was 4.9%-15.9% and 8.0%-20.5% higher than that of W-N₃ and W-N₄ treatments. Meanwhile, the W-G-N₃ treatment improved grain quality of wheat by increasing protein content, sedimentation value, and wet gluten content, among which, the protein content, sedimentation value, and wet gluten content of W-G-N₃ were increased by 12.3%-16.1%, 28.7%-47.2%, and 10.7%-11.1%, respectively, compared with W-N₃; The protein content of W-G-N₃ was increased by 8.9%-12.4% compared with W-N₄, but the differences in sedimentation value and wet gluten content between W-G-N₃ and W-N₄ were not significant. In addition, the W-G-N₃ treatment was beneficial to promote nitrogen uptake and conversion to grain yield in wheat compared with W-N₃ and W-N₄ treatments, in which the N uptake was increased by 42.2%-58.9% and 35.2%-45.0%, N use efficiency was increased by 12.0%-20.6% and 5.9%-20.4%, respectively, and N partial factor productivity was increased by 3.6%-18.3% and 28.1%-58.1%, respectively. The W-G-N₃ treatment could compensate for the reduction of N agronomic efficiency, which was 74.2%-80.0% higher than W-G-N₄ treatment. The correlation analysis showed that multiple green manure after wheat combined with moderate reduction of N fertilizer increased grain yield by promoting efficient nitrogen uptake and utilization, and also significantly improved grain nutritional quality. 【Conclusion】 The combination of multiple green manure after wheat with 85% (153 kg·hm^-2) nitrogen application is the suitable cropping pattern and nitrogen application level to boost wheat yield, improve wheat grain quality, and increase nitrogen use efficiency in Hexi oasis irrigated areas.

Effects of Varied Rapeseed Varieties and Cultivation Measures on Harvest Index

PENG WenLi, WANG Rui, CHEN XiaoLei, LIU AHui, ZHENG WeiDong

Scientia Agricultura Sinica. 2023, 56(17):  3331-3346.  doi:10.3864/j.issn.0578-1752.2023.17.008

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【Objective】 This study aimed to investigate the relationship between harvest index (HI) and dry weight of different organs such as silique wall and seed yield at maturity stage under different rapeseed varieties and cultivation measures, analyze the effects of breeding age, plant height and winter characteristics of rapeseed varieties on HI, and to provide the references for breeding high-yield and high-efficiency rapeseed varieties and coordination and optimization of cultivation measures. 【Method】 Taking 60 rapeseed varieties with different breeding ages, plant heights and winter characteristics popularized in the Yangtze River Basin in recent years as experimental materials, field tests were conducted in Hubei and Guizhou provinces in 2016-2017. In 2017-2020, combinations of different agronomic measures such as sowing date, fertility level, planting density and row spacing configuration were designed, and field experiments were carried out in Bijiang, Sinan and Jiangkou, Guizhou, yield and dry weight of each part were measured after sampling at maturity stage of rapeseed, and harvest index was calculated and analyzed. 【Result】 The difference of HI among different rapeseed varieties was up to 367%, and the difference of HI among different agronomic cultivation measures was 52%-117%. For each 0.1 increase in HI of different rapeseed varieties, seed yield increased by 305 and 385 kg·hm^-2 at two experimental sites, respectively. With the optimization of different agronomic measures, seed yield increased with the increase of rapeseed HI. For every 0.1 increase in HI, seed yield increased by 505-1 690 kg·hm^-2. Under different varieties and cultivation measures, HI was positively correlated with seed yield of main stem, seed yield of branch and aboveground biomass yield. There was a positive correlation between HI and pod dry weight for different optimized cultivation measures. However, there was no consistent correlation between HI and main stem dry weight of rapeseed under different varieties and cultivation measures. There was a significant difference in HI between rapeseed varieties bred before 2010 and after 2015; the HI of rapeseed varieties with plant height less than 170 cm was relatively high, and the HI of higher plant height varieties was significantly less than that of short and medium plant height varieties. There was no significant difference in HI among weak winter, semi-winter and winter rapeseed varieties. 【Conclusion】 Different rapeseed varieties and agronomic measures had significant effects on HI. Varieties with high HI could be selected and popularized. The HI of field crop population should be appropriately increased through optimized and integrated cultivation technology and agronomic measures. Seed yield should be improved through the coordination of good varieties and good measures. In recent years, the HI of rapeseed variety bred and popularized in the Yangtze River basin had been significantly affected by its breeding age and plant height, while the difference in wintering among varieties has no significant impact on HI.

PLANT PROTECTION

Dominated Species and the Predation Assessment of Natural Enemies on Thrips in Cotton Fields in Xinjiang

DANG WenWen, LIU Bing, CHU Dong, LU YanHui

Scientia Agricultura Sinica. 2023, 56(17):  3347-3357.  doi:10.3864/j.issn.0578-1752.2023.17.009

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【Objective】 Thrips is a major pest in cotton fields in Xinjiang. The objective of this study is to select their dominated predatory natural enemies and assess the predation efficiency on thrips, and to provide scientific insights for the development of biocontrol on thrips in cotton fields. 【Method】 The population dynamics of thrips and their predators in Korla cotton field in southern Xinjiang were systematically investigated by visual inspection within five-point sampling in different blocks from 2021 to 2022. Predators’ samples were collected at different growth stages of cotton fields and detected by molecular detection, DNA of the whole natural enemy was extracted first, and PCR amplification was performed using the specific primers of Thrips tabaci and Frankliniella intonsa to obtain the species information of thrips in various natural enemies, then the quantitative food web of predator-thrips was constructed, and the differences of related food web structure at different growth stages of cotton were compared. In addition, basing on the predator-prey functional response model, the predation function of different predators (including adults and juveniles) on thrips was evaluated in laboratory by setting the prey density of 50, 100, 200, 300 and 400 individuals per Petri dish. 【Result】 The population density of thrips was low at the seedling and budding stages but was high at the flowering-bolling stage. Annual population peaks occurred from late-July to early-August. Basing on the population dynamic, Hippodamia variegata is the dominated predator for thrips that occurred across the seeding, budding, flowering and bolling stages, while Campylomma diversicornis is the dominated predator in the late of flowering-bolling stage. The results of molecular detection showed the detection rate of thrips was high in the gut of H. variegata in the whole growth period of cotton, the detection rate of thrips in the gut of Coccinella undecimpunctata at seeding-budding stage was high, and the detection rate in the gut of C. diversicornis was also high at flowering-bolling stage. The predatory function response in laboratory indicated the predation ability of different predators on F. intonsa increased with the increase of prey density, the predation function of the larvae of H. variegata, the nymph of C. diversicornis and the larvae of Chrysoperla carnea was consisted with the Holling-Ⅱ model, whereas the predation function of the adult, such as H. variegata and C. diversicornis was suited with the Holling-Ⅲ model; when high prey density of 400 individuals thrips per Petri dish was provided, the consumed number of thrips for the adult of H. variegata was significantly higher than the larvae, the adult and nymph of C. diversicornis and the larvae of C. carnea.【Conclusion】H. variegata is the dominant predator for thrips across the whole cotton growth period, and C. diversicornis is the dominant predator in the late of flowering-bolling stage. This study provides a scientific insight for the utilization of predators to suppress thrips in cotton fields in Xinjiang.

Selection Preference and Adaptability of Bactrocera dorsalis to Different Varieties of Malus pumila

LI XingXing, ZHOU GuoFu, LUO GuanYu, CHEN SiRong, ZHANG JinLong, CHEN GuoHua, ZHANG XiaoMing

Scientia Agricultura Sinica. 2023, 56(17):  3358-3371.  doi:10.3864/j.issn.0578-1752.2023.17.010

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【Objective】 This study aims to investigate the preference of Bactrocera dorsalis for fruits of six main Malus pumila varieties and to understand the effects of different varieties of M. pumila on the growth and development of B. dorsalis. 【Method】 Three early maturing varieties (Huashuo, Honglu, Gala) and three late maturing varieties (Duanzhifushi, Yanfu No. 3, Qincui) were used as test materials. The hardness and sugar content of M. pumila fruit were determined by hardness tester and a brix refractometer. Three types of fruit handling methods were employed: whole fruit, peeled fruit and fruit odor. Fruits of the same ripening batch of the same handling method were placed in rearing cages, to measure the numbers of visits and laid eggs by B. dorsalis on fruits of different varieties were determined. Feeding different varieties of fruits to the first hatching larvae, the growth and development parameters of larval duration, pupation rate, pupal weight and other parameters after feeding were determined. The selection preference and development adaptability of B. dorsalis to different varieties of M. pumila were analyzed. 【Result】 There were differences in the numbers of visits and laid eggs of B. dorsalis on different varieties of fruits. The female adults were more sensitive to different varieties and fruit handling methods. B. dorsalis preferred to choose Gala among whole fruits of the early maturing varieties, which had 9.0 female visits and 105.0 eggs per fruit, respectively. While among the late maturing varieties, B. dorsalis showed a stronger preference for Qincui, and the numbers of female visits and laid eggs on this variety reached to 5.5 and 64.2 eggs per fruit, respectively. The fruit handling methods influenced the selection preference of B. dorsalis. With the exception of Gala, the numbers of visits and laid eggs on peeled fruits were higher in different fruit handling methods of the same variety. Among the early maturing varieties, Huashuo had held the highest number of laid eggs on the peeled fruit, reached to 201.6 eggs per fruit, and among the late maturing varieties, Yanfu No. 3 had held the highest number of laid eggs on fruit odor, reached to 83.8 eggs per device. Two-way ANOVA found that variety and fruit handling method had an impact on the number of visits and laying eggs. Larvae of B. dorsalis had held the shortest period of 11.9 d in Gala and the shortest pupal stage in Huashuo of 9.9 d after feeding on different fruits. Pupation and emergence rates were higher in late maturing varieties. The difference in the age-stage-specific survival rate of the larvae that fed on different varieties was mainly in the 2nd instar stage, and the 2nd instar larvae which fed on Huashuo and Gala had held fastest development rate and peaked at the 3rd day. 【Conclusion】 There were differences in the selection preference of B. dorsalis on different M. pumila varieties. Gala was preferred in early maturing varieties, while Qincui was preferred in late maturing varieties. Fruit integrity and fruit odor had an effect on the selection preference. The numbers of visits and laid eggs on peeled fruit and fruit odor were generally higher than those on whole fruit. B. dorsalis can finish the growth and development of larvae on M. pumila fruit, and it has the ability to reproduce damage in apple orchards. The correlation between the preference of B. dorsalis for M. pumila fruit and the fitness of offspring’s growth and development is not strong, which is different from the hypothesis of “selectivity and fitness”. In the comprehensive management of B. dorsalis in apple orchards, the damage monitoring of Gala and Qincui varieties should be strengthened, and the damaged fruits should be avoided and removed in time in agricultural operations.

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

The Accumulation of Iron and Manganese in Wheat and Its Relationship with Soil Nutrients Under Long-Term Application of Nitrogen Fertilizer

LIN JiangYun, YIN BenSu, WANG XingShu, LIU ChenRui, SUN Qing, XIE XingXing, CHENG LingLing, SUN LiWei, SHI Mei, WANG ZhaoHui

Scientia Agricultura Sinica. 2023, 56(17):  3372-3382.  doi:10.3864/j.issn.0578-1752.2023.17.011

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【Objective】 The objective of the study was to explore the uptake and distribution of iron (Fe) and manganese (Mn) under long-term application of nitrogen fertilizer and its relationship with soil nutrients, to seek the nitrogen fertilizer regulation strategy based on yield and wheat Fe and Mn nutrition, and to provide a new perspective for the optimization of nitrogen fertilizer application and improvement of wheat quality. 【Method】 Based on the long-term nitrogen fertilizer experiment of winter wheat in dryland, the concentration of Fe and Mn in wheat grains, and their relationship with wheat grain yield, the uptake and distribution of Fe and Mn in wheat and soil nutrients were analyzed under nitrogen application rates of 0, 80, 160, 240, and 320 kg·hm^-2, respectively. 【Result】 Compared with the control, the application of nitrogen improved wheat grain yield, shoot Fe concentration, nutrient Fe and Fe concentration in grains, but decreased Mn concentration in wheat shoot and grains. When the nitrogen application was higher than 160 kg·hm^-2, no differences among treatments were found in the grain yield (5 857-6 598 kg·hm^-2) and grain Fe concentration (40.2-42.2 mg·kg^-1), and the Mn concentration in grains remained at a lower level (30.4-35.3 mg·kg^-1). N application significantly decreased soil pH and increased the proportion of weekly bound organic Fe and Mn and Mn oxide-bonded Fe in the soil, further enriching soil DTPA-Fe and DTPA-Mn content. Meanwhile, the correlation analysis showed that the concentration of Fe in grains had a significantly positive correlation with the yield, biomass, harvest index, spike number, grain number per panicle, grain Fe uptake and Fe harvest index, and soil Mn-oxide-bonded Fe, but it has a significantly negative correlation with the grain Mn concentration and soil residual Fe. However, the grain Mn concentration was significantly negatively correlated with grain yield, biomass, harvest index, spike number, grain number per panicle, grain Fe uptake and Fe harvest index, and was significantly positively correlated with the shoot Mn uptake, but there was no significant association found with various forms of Mn in soil. 【Conclusion】 In the northwest arid area, the long-term nitrogen application on calcareous soil could improve the availability of soil Fe, promote the absorption and localization of Fe in grains, and increase the Fe concentration in grains. Nitrogen application increased the soil Mn availability, but significantly inhibited the uptake of Mn by wheat, and resulted in a significant decrease of Mn concentration in grains. In addition, when the phosphorus supply level is consistent, a long-term lack of soil nitrogen supply may lead to low Fe concentration and high Mn concentration in wheat grains. Considering the yield, the bioavailability of Fe and Mn in grains and the environmentally-economic benefits, it was recommended that nitrogen application rate should be controlled at 160 kg N·hm^-2 on calcareous soil, since excessive nitrogen application fertilizer would not be conducive to further increase grain yield and improve its nutritional quality.

Effects of Supplemental Irrigation After Regreening and Nitrogen Fertilizer Application Rates on Wheat Yield, Water and Nitrogen Use Efficiency in Dryland

ZHAO KaiNan, WU JinZhi, HUANG Ming, LI YouJun, WANG HongTao, HUANG XiuLi, WU ShanWei, ZHANG Jun, ZHAO ZhiMing, ZHAO WenXin, LI ShuJing, LI Shuang, LI WenNa

Scientia Agricultura Sinica. 2023, 56(17):  3383-3398.  doi:10.3864/j.issn.0578-1752.2023.17.012

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【Objective】 The project of high standard farmland construction in China has improved the irrigation conditions in dryland, however, there is usually only once limited irrigation that could be carried out in wheat growing season. In this study, the effects of supplemental irrigation after regreening and nitrogen (N) fertilizer rates on productivity, water and N utilization in dryland wheat and nitrate residue in soil were investigated, so as to provide the theoretical basis and technical support for high-yield, high-efficiency and environment-friendly production in dryland wheat. 【Method】 The two-factor split-plot field experiment was conducted in the typical dryland of the intersection between Loess Plateau and Huang-Huai-Hai Plain from 2019 to 2022, where the irrigation conditions of no-irrigation during whole growth period (I0) and supplemental irrigation after regreening (I1, supplemental irrigation to 85% of field capacity when the soil water content in the 0-40 cm soil layer after wheat regreening was lower than 60% of field capacity at the first time) were assigned to the main plots, and the nitrogen application rates of 0 (N0), 120 kg N·hm^-2 (N120), 180 kg N·hm^-2 (N180) and 240 kg N·hm^-2 (N240) were assigned to the subplots. The grain yield and yield components, water use efficiency, N absorption and utilization in wheat were tested, as well as the nitrate residue in the 0-200 cm soil layer in dryland. 【Result】 Compared with I0, I1 could optimize wheat yield components and increase N absorption capacity, for the 3-year average grain yield and water use efficiency significantly increased by 55.8% and 34.7%, respectively, however, it decreased soil nitrate residue by 11.6% in the 0-200 cm soil layer. With the increase of N application rate, the grain yield, spike number, kernels per spike and water use efficiency initially increased and then decreased, and reached the maximum under the N180 treatment with I0 level, but there were initially increased and then remained stable, and reached the higher level both in the N180 and N240 treatments under I1 level. With the increase of N application rate, the N use efficiency, N uptake efficiency and N partial factor productivity were significantly decreased under the both two irrigation conditions, but the N agronomy efficiency and N apparent efficiency under N180 treatment was not significantly decreased compared with N120 treatment (except I1 level in 2021-2022). Regardless of irrigation, with the increase of N application rate, the soil nitrate residue at harvest were significantly increased. Soil nitrate accumulated largely in the 0-60 cm soil layer under each N application rates with I0 level, and the obvious accumulated effects of soil nitrate residue in the 0-200 cm soil layer were found under N180 and N240 treatments; meanwhile, the soil nitrate residue under I1 level in the 0-60 cm soil layer was significantly lower than that under I0, and there were only accumulated effects of soil nitrate residue was found in the 0-200 cm soil layer under N240 treatment. In general, both the I1N180 and I1N240 could obtain the higher grain yield and water use efficiency. I1N180 treatment reduced water consumption during growth period and soil nitrate residue in the 0-200 cm soil layer, and improved the N efficiency, compared with I1N240 treatment. 【Conclusion】 Supplemental irrigation after regreening combined with 180 kg N·hm^-2 N fertilizer could optimize the yield components, shoot N accumulation and N accumulative rate, thus it could not only improve the grain yield and N absorption and utilization efficiency in wheat, but also reduce the soil nitrate residue in 0-200 cm soil layer. Therefore, I1N180 treatment was an optimal model for realizing the collaborative target of high-yield, high-efficiency and environment-friendly in dryland wheat production system.

Spatial Distribution of Nitrate in Vineyards Soils in Yongding River Basin, Hebei Province

LI SiQi, WANG ZhiHui, CHANG YuYao, JI YanZhi, GUO YanJie, LIU Jun, ZHANG LiJuan, WANG YaJing

Scientia Agricultura Sinica. 2023, 56(17):  3399-3411.  doi:10.3864/j.issn.0578-1752.2023.17.013

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【Objective】 The relationship between nitrogen input, elevation of vineyards and soil nitrate content and accumulation in Yongding River Basin was studied, in order to provide a theoretical basis for rational fertilization and reduction of environmental pollution risks of vineyards in Yongding River Basin. 【Method】 52 typical vineyards in Yongding River Basin of Hebei Province were selected as the research objects. The current situation of nutrient input in the vineyard was analyzed through field investigation. Soil nitrate contents of 0-60 cm (20 cm interval) was measured through indoor analysis, and its accumulation and surplus were calculated. Spatial variability of nitrogen input and surplus, soil nitrate content and accumulation were analyzed by ArcGIS geostatistics. 【Result】 Less than 50% of farmers in Yongding River basin applied organic fertilizer, mainly using inorganic fertilizer. The average nitrogen input in upstream and downstream vineyards was (1 492.79±988.90) and (1 079.31±638.25) kg·hm^-2, respectively. The average nitrogen surplus were (1 430.41±993.01) and (1 027.23±637.37) kg·hm^-2, respectively. There was a significant positive correlation between nitrogen input and surplus (P<0.01), and the spatial distribution showed a decreasing trend from west to east. The variation and spatial distribution of soil nitrate content and accumulation in different soil layers were consistent. The low value area was mainly distributed in the downstream, while the high value area was mainly distributed in the upstream. The average nitrate content of 0-60 cm soil profile in the upper and lower reaches was 34.96 and 18.76 mg·kg^-1, respectively, and the average cumulative amount was 92.44 and 48.12 kg·hm^-2, respectively, which showed significant differences among different soil layers. Soil nitrate content and accumulation in the upper reaches were the lowest in the 20-40 cm soil layer, and increased with the increase of soil layer in the lower reaches. Soil nitrate content and accumulation in the upper reaches were the highest at 600-650 m elevation, which were significantly higher than those at other elevations (P<0.05). However, soil nitrate content and accumulation in the lower reaches were not significantly affected by elevation. Correlation analysis showed that the distribution of nitrate accumulation in the surface layer were mainly affected by elevation, while in the bottom layer were mainly affected by nitrogen input. 【Conclusion】 In the Yongding River Basin, the nitrogen surplus of vineyards in the study area was serious, and the nitrate in the vertical soil layer accumulated to the deep layer. The soil nitrate content and accumulation at different elevations (except 450-500 m) were higher in the upstream than in the downstream, but the variation trend of vertical distribution was different, which was jointly affected by the elevation and nitrogen input.

HORTICULTURE

Effects of Different Distances from Original Planting Row on Tree Growth and Fruit Yield of Young Trees of G935 Dwarf Rootstock Miyato Fuji Under Continuous Cropping

LI MinJi, LI XingLiang, ZHANG Qiang, ZHOU Jia, YANG YuZhang, ZHOU BeiBei, ZHANG JunKe, WEI QinPing

Scientia Agricultura Sinica. 2023, 56(17):  3412-3419.  doi:10.3864/j.issn.0578-1752.2023.17.014

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【Objective】 The effects of five different planting distances from the original planting line on the growth of young apple trees of G935 dwarf self-heeling rootstock Miyato Fuji were investigated and studied for four consecutive years, and the resistance to repeated cropping of G935 dwarf self-heeling rootstock grafted Miyato Fuji was evaluated, so as to provide a theoretical basis for the renewal of old and inefficient apple orchards and the upgrading of cultivation models in China. 【Method】 In the spring of 2018, the 12-year-old apple tree (Miyato Fuji/SH6/seedling stock) was planted, without soil sterilization, adding organic fertilizer, chemical fertilizer and biological microbial fertilizer, and the G935 dwarf self-rooting stock Miyato Fuji seedlings (2-year root and 1-year dry) were directly planted at different distances (0, 0.5, 1, 1.5 and 2 m) from the original planting line, and the fine spinning hammer tree shape was used for pruning. The differences in tree growth, leaf function, early flowering and early fruiting, and fruit yield and quality of young trees of G935 dwarf self-rooting rootstock Miyato Fuji were investigated under 5 treatments for 4 consecutive years after planting. 【Result】 Under the condition of replanting, there was no significant difference in the growth, leaf function, early flowering and early fruiting, and fruit yield and quality of G935 dwarf self-rooting rootstock Miyato Fuji at different distances from the original planting line. Within 4 years after replanting, with the growth of tree age, the height, trunk diameter and number of main branches of Fuji trees under five treatments increased year by year. In the fourth year after replanting, the average number of main branches in each treatment reached more than 30. From the second year of planting, the proportion of long branches under each treatment decreased year by year, and the proportion of short branches increased year by year. In the fourth year of continuous cropping, there was no significant difference in the growth of new shoots, chlorophyll content of leaves, net photosynthetic rate and one hundred leaves weight (fresh weight and dry weight) of Fuji trees under different treatments; the average yield per plant and fruit quality (average fruit weight, fruit shape index, titratable acid content, soluble solid content, and fruit solid-acid ratio) of Fuji fruit under all treatments were similar, without significant difference. 【Conclusion】 Under the condition of replanting in continuous cropping, there was no significant difference in the growth, leaf function, early flowering and early fruiting, fruit yield and quality of the young trees planted with G935 dwarf self-rooting rootstock Miyato Fuji at different distances from the original planting line four years before planting. The branch composition of the trees in each treatment was reasonable, the tree vigor was moderate and not weak, the flowering was early, and the fruit quality was good. G935 was suitable for replanting in continuous cropping, and the effect of resistance to repeated cropping was not affected by the distance from the original planting line.

Genetic Variation Analysis and Candidate Genes Mining of Regulating Flesh Color in Pomelo

CHENG Li, YANG ShengNan, ZHU YanSong, WANG Xu, ZHAO WanTong, LI RenJing, LI Pei, YUAN ZhongJie, JIANG Dong

Scientia Agricultura Sinica. 2023, 56(17):  3420-3434.  doi:10.3864/j.issn.0578-1752.2023.17.015

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【Objective】 The flesh color is an important appearance and quality trait of pomelo varieties. The aim of this study was to excavate variation loci and genes significantly related to the flesh color of pomelo varieties, so as to lay a foundation for further understanding the flesh color development mechanism and marker-assisted breeding of pomelo varieties. 【Method】 100 pomelo accessions were used to identify their flesh color with a color difference meter, and classified based on their phenotype results. The simplified genome sequencing of 100 pomelo accessions was carried out using GBS (genotyping-by-sequencing) technology. The eigenvalues and eigenvectors of the genotype data obtained from sequencing were calculated by GCTA software to analyze the population structure, and the genetic differentiation index (Fst) of two different flesh color populations were calculated using Plink 2.0 software. The GLM model in GEMMA software was adopted to perform genome-wide association analysis, and the variation loci significantly associated with color phenotype were identified for further allelic variation analysis. According to the citrus linkage disequilibrium (LD) distance, the candidate genes located in the 25 kb adjacent region around the variation loci that might be related to the forming of flesh color were screened out, four white and four red pomelo accessions were randomly selected to perform qPCR to obtain the candidate genes expression at different fruit development stages. 【Result】 According to the phenotype of flesh color, 100 accessions were divided into two categories corresponding to white and red pomelo accessions, including 58 white flesh pomelos and 42 red flesh pomelos. Six SNP sites with Fst index greater than 0.4 and -log₁₀ (P)>9 were screened out after Fst genetic differentiation analysis and GWAS genome-wide association analysis. The genotypes of six variation SNPs sites were extracted from 100 pomelos accessions and the genotypes of these SNPs could be used to predict the flesh color of pomelos. A total of 14 genes that function annotated as isoprene synthesis, plastid development, abscisic acid signal regulation, ethylene response related genes and transcription factors were screened and selected. Genes expression analysis showed that Cg7g013760 (mitogen-activated protein kinase kinase MAPKKK17), Cg7g013840 (chloroplast porphyrinogen deaminase), Cg7g014020 (transcription factor TCP7), Cg7g014120 (chloroplast superoxide dismutase SOD3), Cg7g014190 (FAD synthetase 2) were related to the forming of flesh color in pomelos. 【Conclusion】 A total of 6 SNP sites significantly associated with flesh color in pomelos accessions were identified by GWAS and Fst analysis. A total of 14 genes within the 25 kb regions around the mutation SNPs site were screened and annotated. The gene expression analysis showed that five genes might be involved in the forming of flesh color in pomelo accessions.

FOOD SCIENCE AND ENGINEERING

Effect of Heat Treatment on the Warmed-Over Flavor of Nanjing Water-Boiled Salted Duck Detected by HS-SPME-GC-MS Technology and Electronic Nose

WU ShiHao, HUANG TianRan, HUANG Ming

Scientia Agricultura Sinica. 2023, 56(17):  3435-3451.  doi:10.3864/j.issn.0578-1752.2023.17.016

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【Objective】 By using flavor detection technology, the effect of heat treatment on Nanjing water-boiled salted duck’s warmed-over flavor (WOF) was investigated, and the main components of warmed-over flavor were identified. The study could fill the gap in research on warmed-over flavor of Nanjing water-boiled salted duck, as well as provide a basis for controlling the odor associated with poultry processing. 【Method】 The raw materials for the Nanjing water-boiled salted duck were 12 cherry valley duck legs, washed, dry pickled, wet pickled, cooled, and boiled. The samples were vacuum packed in high-temperature retort pouches and randomly divided into four groups after cooling. In order to examine the changes in volatile odor substances in water-boiled salted ducks after heat treatment, one group was used as a control without heat treatment, and the other three groups were heated at different temperatures (80 ℃ 50 min, 100 ℃ 30 min, and 121 ℃ 15 min). By using HS-SPME-GC-MS in combination with electronic nose and sensory evaluation, the effect of heat treatment on Nanjing water-boiled salted duck's warmed-over flavor was studied by cluster analysis and PLS-DA. 【Result】 Under different heat treatment conditions, Nanjing water-boiled salted duck contained different volatile flavor substances. Sensory evaluation showed that the highest warmed-over flavor was found in the 121 ℃ heat treatment group, followed by 100 ℃ and 80 ℃ group, and the best flavor was found in water-boiled salted duck without heat treatment. A total of 78 flavor compounds were detected in the four groups, mainly including aldehydes, ketones, hydrocarbons, alcohols, nitrogenous sulfur, and benzene. These 78 flavor substances were analyzed for OAV, and 22 active odors were detected with OAV>1, among these 22 active odor substances, cluster analysis showed that Valeraldehyde, 2-Heptanone, Decanal, Dodecanal, Octanal, Hexanal, Heptanal, Nonanal, 2,5-Octanedione, 1-Octen-3-ol and 2-Pentylfuran were the most abundant in the 121 ℃ group. Nonanal, Octanal, Valeraldehyde, 1-Octen-3-ol, and 2-Pentylfuran were found to have VIP>1 and the highest content at 121 ℃ in the OPLS-DA analysis (P<0.05). 【Conclusion】 It has been found that heat treatment at 121 ℃ increased oxidative degradation of lipids in water-boiled salted duck, significantly reduced the content of representative aroma substances, and increased the amount of representative odor substances. Nanjing water-boiled salted duck’s warmed-over flavor mainly consisted of Nonanal, Octanal, Valeraldehyde, 1-Octen-3-ol, and 2-Pentylfuran.

ANIMAL SCIENCE·VETERINARY SCIENCE

Establishment of Quantization Method and Genetic Basis Analysis of Brown Eggshell Color in the Lüeyang Black-Boned Chicken

CHEN Qiu, HUANG JingJing, WANG ZhePeng

Scientia Agricultura Sinica. 2023, 56(17):  3452-3460.  doi:10.3864/j.issn.0578-1752.2023.17.017

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【Background】 Brown eggshell color is closely relevant to eggshell strength, concentrations of egg white antimicrobial protein and yolk carotenoid, blood and meat spots, and hatchability, and is an important index affecting quality and sale of eggs. However, due to absence of selection for eggshell color, color of brown eggs that some indigenous breeds lay is light and highly variable, which has an adverse effect on sale of eggs and creation of egg brands. 【Objective】 The aims of this study are to establish a quantization method that could accurately and sensitively capture the variation of eggshell color, to estimate breed-specific heritability of eggshell color and to identify candidate genes associated with eggshell color in the Lüeyang black-boned chicken (LBC), so as to provide a theoretic basis for genetic improvement of LBC eggshell color. 【Method】 841 hens from 62 half-sibling families of LBC breeding population were selected. Three eggs were collected from each hen, and then the eggshell color was quantified using the L*a*b color space, and posterior heritabilities of L, a, and b values were estimated in a Bayesian framework using the Markov Chain Monte Carlo (MCMC) algorithm. The prior distributions of breeding value and residual variances were set using an inverse-Gamma distribution. MCMC performed 130 000 iterations, dropped 30 000 iterations at the beginning and stored one every 100 iterations to obtain posterior distributions of variance components and posterior heritability estimates of color indexes. Hens that laid light brown (n=8), brown (n=8) and light blue (n=8) eggs were selected from LBC population. Three eggs were collected from each hen for the measurement of eggshell pigment, and the absorbance values were measured at 670 nm for biliverdin and at 412 nm for protoporphyrin. The concentrations of protoporphyrin and biliverdin were calculated by regression equations, which were fitted using absorbances and concentrations of standard samples. Shell glands of brown- (n=8) and light brown-shelled (n=8) hens were collected. Expression levels of ALAS1 and CPOX in shell glands were detected using qPCR. 【Result】 Among three color indexes, a* value kept a high consistency with the change of eggshell color based on subjective perception as a* value generally decreased with transition of egg color from brown to green hues. L and b values could accurately reflect the change of egg color from light to dark brown, but L and b values were unable to discriminate between brown and green hues reliably. L and b values showed low variation as L and b values of 50% of samples were distributed between 73.3-79.1 and 13.1-18.2. In contrast, a* values were evenly distributed among the samples as a* values of 50% of samples ranged from 1.4 to 7.1. In line with the distribution characteristics, the coefficient of variation (88.2%) of a value was higher than ones of L (5.7%) and b (24.0%) values. Estimation results of heritability showed that a* value (h²=0.77) was predominately affected by genetic factors. In contrast, L (h²=0.46) and b (h²=0.37) values were controlled by environmental effect to larger extents. For the relationship of eggshell pigment and color, protoporphyrin concentration had a strong correlation with all of three color indexes (L: r=-0.86, a: r=0.73, b: r=0.88). But biliverdin concentration showed a strong (r=-0.73) negative correlation with a* value alone. ALAS1 and CPOX were two key enzymes that catalyze the biosynthensis of protoporphyrin precursors. Expression results showed that expression levels of CPOX in shell glands of brown-shelled chickens were 1.5-fold higher (P<0.05) than that in shell glands of light brown-shelled chickens. ALAS1 had no significant (P>0.05) difference between brown- and light brown-shelled chickens. 【Conclusion】 These results indicated that protoporphyrin was the key pigment affecting LBC eggshell color, and CPOX was a candidate associated with protoporphyrin concentration and color of brown eggs. The a* value was an optimal index quantifying eggshell color of LBC with high accuracy and sensitivity. In view of high heritability estimate of a* value, it was possible to increase brown hue and uniformity of LBC eggshell color via positive selection of a* value.

Prokaryotic Expression and Metal Binding Characterization of Metallothionein 1A and 2A of Sus scrofa

YANG HuiZhen, YANG Huan, WU ZiXuan, FAN KuoHai, YIN Wei, SUN PanPan, ZHONG Jia, SUN Na, LI HongQuan

Scientia Agricultura Sinica. 2023, 56(17):  3461-3478.  doi:10.3864/j.issn.0578-1752.2023.17.018

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【Objective】 The study mainly made the bioinformatics analysis, carried out prokaryotic expression, and explored the metal binding characterization of Sus scrofa metallothionein-1A (SsMT-1A) and metallothionein-2A (SsMT-2A) with Zn(Ⅱ) and Cu(Ⅰ), so as to provide a theoretical basis for the study of the mechanism of action of adding Zn and Cu in diet inducing porcine metallothionein expression, thereby regulating the Zn and Cu homeostasis in vivo, and then promoting the porcine production performance. 【Method】 Firstly, the porcine gene sequences of SsMT-1A and SsMT-2A were obtained from NCBI. Afterwards, their protein sequence characteristics were analyzed by ClustalX2 software and ExPASy database, and then the phylogenetic tree of MT protein molecular was constructed between them and other species using the Mega-X. Secondly, the prokaryotic expression vector pET-28a-SUMO-SsMT-1A/SsMT-2A were constructed and verified by PCR, double digestion, and gene sequencing. Subsequently, the recombinant plasmid was transformed into BL21(DE3) plysS and the expression of SsMT-1A and SsMT-2A was induced using the IPTG. Then, the SUMO-SsMT-1A/SsMT-2A recombinant protein were purified by Ni-NTA affinity chromatography and Superdex-75 column, and were analyzed by Western-blot. In the end, the properties of SsMT-1A and SsMT-2A binding Zn(Ⅱ) and Cu(Ⅰ) were surveyed by the tolerance analysis of Escherichia coli containing SsMT-1A and SsMT-2A genes, circular dichroism (CD), matrix assisted laser analytic ionization time of flight mass spectrometry (MALDI-TOF-MS), and isothermal micrometer calorimetry (ITC). 【Result】 Bioinformatics analysis showed that the protein sequences of SsMT-1A and SsMT-2A were highly homologous, and their cysteine (Cys) content and arrangement motifs were perfectly consistent, and there existed only 8 amino acid residues discrepancy. Through the prokaryotic expression, purification, and SUMO enzyme digestion, the SsMT-1A/SsMT-2A fusion protein were successfully obtained. The tolerance analysis of metal demonstrated that compared with SsMT-2A, E. coli containing SsMT-1A genes had the stronger resistance to Zn and Cu. The CD spectrum illustrated that SsMT-1A and SsMT-2A could combine with Zn(Ⅱ) and Cu(Ⅰ) and both of them exhibited the preference with Zn(Ⅱ) coordination. But SsMT-1A exhibited the stronger binding ability with Zn(Ⅱ) than SsMT-2A. MALDI-TOF-MS results showed that the experimental molecular weights of apo-SsMT-1A and apo-SsMT-2A were respective 6 047.5 Da and 6 048 Da, and the stability order of both of them was and Cu(Ⅰ)> Zn(Ⅱ). ITC results showed that the affinity constants of SsMT-1A and SsMT-2A coordinating Cu(Ⅰ) were higher than that of Zn(Ⅱ), and the stoichiometries of both of them binding Cu(Ⅰ) were 7, while the stoichiometry of SsMT-1A binding Zn(Ⅱ) was 2. 【Conclusion】 Although SsMT-1A and SsMT-2A possessed the high homology, the difference of 8 amino acid residues determined their different binding feature with Zn(Ⅱ) and Cu(Ⅰ). Although SsMT-1A and SsMT-2A shared a highly consistent binding behavior of Zn(Ⅱ) and Cu(Ⅰ), their characteristics of binding Zn(Ⅱ) and Cu(Ⅰ) was slightly different. Thus, they should not be considered as completely physiological equivalent molecules. According to the relationship of structure and function of MT, SsMT-1A might play a role in detoxification of heavy metal ions and SsMT-2A might mainly regulate Zn homeostasis. The study laid a foundation for further elucidating the role of SsMT-1A and SsMT-2A in regulating metal ion homeostasis to promote pig production performance.

Construction of Infectious cDNA Clone of GETV SC483 Strain

DU BingChen, WANG Ming, LIU ChunGuo, WANG ShiDa, WEI XinYu, LU YaMan, SUN ZhenZhao, LIU ZaiSi, WEI LiLi, WANG JingFei, YANG DeCheng

Scientia Agricultura Sinica. 2023, 56(17):  3479-3486.  doi:10.3864/j.issn.0578-1752.2023.17.019

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【Background】 Getah virus (GETV) is a mosquito-borne arbovirus, and belongs to the Alphavirus genus in the Togaviridae family. GETV has an extensive host range, which includes multiple mammals, such as pig, horse, cattle, fox and so on, although GETV is not known to cause disease in human. GETV infection often causes clinical symptoms, such as fever, urticarial rash, arthritis, reproductive disorders, and fetal death. GETV is widely prevalent in the world, and the prevalence in China is on the rise in recent years. In 2018, it suddenly broke out in many pig farms in southern China. The harm for livestock and poultry production and public health caused by GETV has got widespread attention. Currently, there is still no specific antiviral drug or vaccine against GETV. Due to the lack of research on GETV, its biological characteristics, pathogenicity, pathogenesis and epidemic properties are largely unknown. 【Objective】 The aim of this study was to develop a reverse genetics platform of GETV, which could provide a basis for investigating the structure and function of GETV genome, the genetic determinant of pathogenesis, and developing novel vaccine against the virus. 【Method】 The full-length genome of the GETV SC483 strain flanked by a hammerhead ribozyme (HamRz) and hepatitis delta ribozyme (HdvRz) sequence was chemically synthesized and cloned into a low-copy plasmid pOK12-CMV to generate the recombinant plasmid pGETV-SC483, which was transfected into BHK-21 cells to rescue the virus. Then, compared with the parental virus, the rescued-virus was serially passaged, identified, and analyzed for biological characteristics. The genetic stability of the recombinant plasmid pGETV-SC483 in E. coli was also verified. 【Result】 The typical cell pathogenic effect (CPE) of GETV was observed in the cells after being transfected with the pGETV-SC483 for 48 h. The rescued GETV, namely rSC483, was successfully obtained, which had a G4332A mutation resulted in the deletion of a Not I restriction site to differentiate from the parental virus. The rescued virus was further verified by indirect immunofluorescence assay using GETV specific antibody as detection antibody and electron microscopic observation of virus particles. Furthermore, plaque phenotype and growth curve of the rescued virus were similar to that of its parental virus in BHK-21 cells. In addition, the infectious cloning plasmid pGETV-SC483 had good genetic stability in E. coli DH5α. 【Conclusion】 The successfully constructed full-length infectious cDNA clone of GETV SC483 strain provided a technical platform for the study of biological characteristics and pathogenesis of GETV as well as development of novel vaccine against GETV.