Scientia Agricultura Sinica

Improvement and Application of the Method for Determining Yellow Pigment Content in Wheat Grain

ZHAI ShengNan,LIU AiFeng,LI FaJi,LIU Cheng,GUO Jun,HAN Ran,ZI Yan,WANG XiaoLu,LÜ YingYing,LIU JianJun

Scientia Agricultura Sinica. 2021, 54(2): 239-247. doi:10.3864/j.issn.0578-1752.2021.02.001

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【Objective】The yellow pigment content (YPC) in wheat grain is an important factor affecting the color and nutritional quality of flour end-use products. To quickly, accurately and simply detect YPC in wheat grain, the traditional method for YPC determination, AACC 14-50, was improved, providing a technical support for genetic improvement of the color and nutritional quality of flour end-use products.【Method】In this study, improvements have been made to the AACC 14-50 method using a Multiskan Spectrum microplate reader. The accuracy and stability of the modified method were analyzed, and the influence of extraction time and preservation time on flour YPC was dissected. The YPC of 283 domestic and foreign wheat varieties were determined by the improved method to further verify its effectiveness and explore excellent resources for genetic improvement of the color and nutritional quality of flour end-use products. 【Result】 The results showed that YPC determined by the modified method was significantly and positively correlated with those by the traditional AACC 14-50 method, with a correlation coefficient of 0.994 (P<0.001), and analysis of variance showed that YPC measured by the two methods was significant among varieties. The YPC of 144 wheat varieties were measured repeatedly using the modified method, and the correlation coefficients of YPC among three replicates were 0.997, 0.998 and 0.998, respectively. The flour YPC of Gaoyou 5218, Jishi 02-2 and Zhengmai 366 reached the maximum value (0.93, 1.24 and 1.53 μg·g^-1) at the extraction time of 30 min, and remained stable during 30-120 min (0.94-0.97 μg·g^-1, 1.30-1.33 μg·g^-1 and 1.59-1.62 μg·g^-1). No significant changes were observed in the flour YPC of Gaoyou 5218, Jishi 02-2 and Zhengmai 366 stored at room temperature for 0-20 days (0.93-0.97 μg·g^-1, 1.29-1.33 μg·g^-1 and 1.60-1.64 μg·g^-1; P>0.05). A wide range of YPC variation was identified in 283 domestic and foreign wheat varieties, with correlation coefficients ranging from 0.73 to 0.98 among different environments. Briefly, the mean YPC of varieties in the Huang-Huai wheat region was 1.15 μg·g^-1, ranging between 0.51-2.42 μg·g^-1. The average value of YPC in varieties from the Northern winter wheat region was 1.57 μg·g^-1, ranging from 0.90 to 2.52 μg·g^-1. The average value of YPC in the Middle-Lower reaches of the Yangtze River wheat region was 1.07 μg·g^-1, and the variation ranged from 0.56 to 2.54 μg·g^-1. The average YPC of foreign varieties was 1.61 μg·g^-1, ranging from 0.94 to 2.48 μg·g^-1. Twenty-four varieties with lower YPC and 26 with higher YPC were identified, which can be used as parents in wheat breeding programs to improve the color and nutritional quality of flour end-use products. Compared with the traditional AACC 14-50 method, the modified one reduces the workload, decreases the detection time, and increases the detection efficiency by about 12-15 times, and the consumption of samples and reagents is only 1/16 of those in the traditional method, which greatly reduces the cost. 【Conclusion】 The modified method for determining YPC in wheat grain established in this study is accurate, reliable, economical, efficient and simple, which can replace the traditional AACC 14-50 method for a large-scale determination of YPC.

Cloning and Functional Analysis of Salt Stress Response Gene GhPEAMT1 in Upland Cotton

WANG Na,ZHAO ZiBo,GAO Qiong,HE ShouPu,MA ChenHui,PENG Zhen,DU XiongMing

Scientia Agricultura Sinica. 2021, 54(2): 248-260. doi:10.3864/j.issn.0578-1752.2021.02.002

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【Objective】Phosphoethanolamine-N-methyltransferase (PEAMT) is the key enzyme in the synthesis of plant phosphocholine, which is the precursor of choline, a glycine betaine synthesis substrate that can enhance plant resistance. The biological function of GhPEAMT1 gene in response to salt stress in upland cotton was studied by cloning, expression pattern analysis and functional verification, so as to provide gene resources for breeding salt tolerant cotton varieties.【Method】According to the screening of transcriptome sequencing data, GhPEAMT1 gene was selected as a salt-tolerant candidate gene. The target gene was amplified by polymerase chain reaction (PCR). Gene structure characteristics predict protein relative molecular mass and evolutionary relationship were analyzed by bioinformatics; Salt-tolerant cotton variety Earlistable 7 and salt-sensitive cotton genotype Nandanbadidahua were treated with 200 mmol·L ^-1 NaCl solution, and cotton leaves and roots were taken for fluorescence quantitative PCR (qRT-PCR) to analyze the expression characteristics in tissues. Subcellular location vector was constructed for transient transformation of tobacco to determine the location of protein in the cell. The gene overexpression vector was constructed, and Arabidopsis thaliana was transformed by inflorescence infection method, and the germination rate and taproot length of transgenic Arabidopsis under salt stress were analyzed. Virus induced gene silencing (VIGS) technology was used on Earlistable 7 to silence the target gene and the efficiency of gene silencing was verified by real-time fluorescence quantitative analysis of the cotton leaves. Then the enzyme activities of catalase (CAT) and glutathione peroxidase (GPX) in the leaves of cotton were measured. 【Result】Two homologous genes, GhPEAMT1A and GhPEAMT1D, were cloned and the CDS lengths were 1 488 bp and 1 485 bp, respectively. The phylogenetic tree showed that GhPEAMT1 had the closest genetic relationship with the Sea island cotton, and had the higher homology with Cocoa compared with other species. Under salt stress conditions, the expression levels of GhPEAMT1A gene and GhPEAMT1D gene in leaves and roots of salt-tolerant Earlistable 7 were significantly higher than that of salt-sensitive Nandanbadidahua. Subcellular localization shows that the gene is located in the cytoplasm. Overexpression of this gene for Arabidopsis thaliana found that the germination rate of transgenic Arabidopsis thaliana was significantly higher than that of wild-type on MS medium of 100 and 150 mmol·L ^-1 NaCl. The taproot root length of transgenic Arabidopsis was significantly longer than that of wild-type on the MS medium of 50 mmol·L ^-1 and 100 mmol·L^-1 NaCl. After treatment with 40 g·L^-1 NaCl solution for 24 h, the cotton leaves with successfully silenced GhPEAMT1A and GhPEAMT1D gene were more wilting than those of CK and TRV2:00 injections. After salt stress, the TRV2::GhPEAMT1A cotton plants were reduced in catalase (CAT) and glutathione peroxidase (GPX) content in comparison with the unloaded injected cotton plants. 【Conclusion】Arabidopsis overexpression and VIGS experiments show that the GhPEAMT1 gene can respond to salt stress and play a positive regulatory role.

Creation of High-Oleic (HO) Canola Germplasm and the Genetic and Physiological Analysis on HO Trait

LONG WeiHua,PU HuiMing,GAO JianQin,HU MaoLong,ZHANG JieFu,CHEN Song

Scientia Agricultura Sinica. 2021, 54(2): 261-270. doi:10.3864/j.issn.0578-1752.2021.02.003

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【Objective】 This study is to create the new high oleic (HO) canola germplasm, to explore its genetic mode and the physiological characters of the HO trait, which will lay a foundation for breeding HO canola varieties. 【Method】 The primary mutation population with was obtained by radiation treatment of germinating canola seeds. The new HO germplasm was screened by extreme selection method combined with microspore culture technology in subsequent generations. The genetic populations of six generations (P₁, P₂, F₁, BC₁P₁, BC₁P₂ and F₂) were constructed by crossing the HO Germplasm with three conventional canola lines with different genetic background. After the fatty acid content of each population was determined, the genetic analysis of high oleic acid content in the genetic population was analyzed by the mixed major-gene plus polygene inheritance model. The oleic acid content in cotyledons during seed germination, vegetative organs at seedling stage in different temperature regimes and seeds during silique ripening process of the HO germplasm were detected to explore their change patterns and physiological effects.【Result】 The primary mutation population with significantly increased oleic acid content was obtained by radiation treatment, and then the high generation population with 20-percent-increased oleic acid content was obtained by using extreme selection method in subsequent generations and the double haploid population was obtained by microspore culture. Finally, a new HO germplasm B161 (C18:1=85%, C18:3=3%) was successfully screened according to the quality traits. Three genetic populations with different genetic background were obtained by crossing B161 as HO parent with three other conventional lines. The correlation analysis of fatty acid contents showed that there was a significant negative correlation between oleic acid content, linoleic acid content and linolenic acid content. The results of genetic analysis showed that the high oleic acid content was controlled by two major genes with additive effect, and their effect values were close. Physiological analysis showed that the contents of oleic acid in vegetative organs (root, stem, leaf and petiole) of HO line were significantly higher than those of the conventional strain, and the linolenic acid contents of HO line were significantly lower than those of the conventional line. The contents of oleic acid in vegetative organs of HO line decreased at low temperature, but they were still higher than those of the conventional line. The linolenic acid contents in vegetative organs of the two lines increased significantly at low temperature, but the linolenic acid content of HO line was still lower than that of the conventional line. During the process of seed ripening and seed germination, the oleic acid content of HO line was significantly higher than that of conventional line, while the linolenic acid content was significantly lower than that of conventional line.【Conclusion】The new HO germplasm was successfully created and the genetic mode and physiological characters were confirmed. This HO germplasm has the potential value in breeding.

Damage of AsA-GSH Cycle of Soybean Leaves Under Waterlogging Stress at Initial Flowing Stage and the Mitigation Effect of Uniconazole

WANG ShiYa,ZHENG DianFeng,XIANG HongTao,FENG NaiJie,LIU Ya,LIU MeiLing,JIN Dan,MOU BaoMin

Scientia Agricultura Sinica. 2021, 54(2): 271-285. doi:10.3864/j.issn.0578-1752.2021.02.004

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【Objective】 The aim of this study was to investigate effects of waterlogging stress on the ascorbate-glutathione (AsA-GSH) cycle of soybean leaves and the regulating effect of uniconazole (S3307) during initial flowering stage (R1), so as to provide a theoretical basis for improving soybean waterlogging resistance and the application of uniconazole.【Method】 This study was conducted in the pot plant of the National Coarse Grain Engineering Technology Research Center of Bayi Agricultural University in Heilongjiang in 2019. The water-tolerant variety KenFeng 14 and the waterlogging-sensitive variety KenFeng 16 were used as test materials for pot planting experiments. The leaves were sprayed with S3307 (concentration 50 mg·L^-1, appropriate spray amount 225 L·hm^-2). After spraying S3307 for 5 days, the waterlogging stress treatment was started after 0 d (R1+5) and 5 d (R1+10) and normal water treatment for 5 days (R1+15) after sampling, respectively, and the various physiological indicators were measured by using spectrophotometer to study the degree of membrane lipid peroxidation (MDA) of soybean leaves under waterlogging stress. The effects of reactive oxygen species (ROS) and non-enzymatic antioxidants (AsA, DHA, GSH, and GSSG) and key enzymes (APX, GR, MDHAR, and DHAR) in the AsA-GSH circulatory system and the mitigating effects of S3307 were analyzed. 【Result】 In the R1 stage after R1+5 of waterlogging stress, compared with CK, the S3307 treatment reduced the MDA, $\mathop{{O}}_{2}^{{\mathop{}_{\ ·}^{-}}}$ production rate and H₂O₂ content, and also increased the content of non-enzyme antioxidants and key enzymes in the AsA-GSH cycle to maintain ROS balance, thus promoted the growth and development of two soybean varieties. At R1+10, W treatment significantly increased the MDA content in the leaves of the two soybean varieties, which were significantly increased by 40.02% and 37.53% higher than that of CK, respectively, and accelerated ROS ($\mathop{{O}}_{2}^{{\mathop{}_{\ ·}^{-}}}$ and H₂O₂); the $\mathop{{O}}_{2}^{{\mathop{}_{\ ·}^{-}}}$ production rate of the two varieties under waterlogging stress was significantly increased by 60.29% and 27.77%, compared with CK, respectively; The H₂O₂ content was significantly increased by 49.45% and 43.40% compared with CK, respectively; The waterlogging sensitive variety KenFeng 16 suffered more damage than the waterlogging resistant variety KenFeng 14, and at the same time, under the waterlogging stress, the antioxidant substances and key enzyme activities in the leaves of both soybean varieties were increased to adapt to the waterlogging stress response from stress. After S3307 treatment, W+S treatment could further increase antioxidant substances (AsA, GSH), redox substances (DHA, GSSG), total ascorbic acid (AsA + DHA) and total glutathione (GSH +GSSG) content under waterlogging stress.content, and increase the activity of antioxidant enzymes (APX, GR, MDHAR, DHAR), reduce the MDA content of the leaves, inhibit the accumulation of ROS, and reduce the damage caused by waterlogging stress to the membrane system. After returning to normal water treatment for 5d (R1 + 15), the above indexes of two soybean varieties W treatment were reduced, but W+S treatment could maintain the two soybean leaves. The higher antioxidant enzyme activity and antioxidant substance content accelerated the removal of excessive accumulation of MDA and ROS, promoted the AsA-GSH cycle operation in soybean leaves under waterlogging stress, and then promoted the return of the two soybean varieties to normal state.【Conclusion】 Waterlogging stress had different degrees of influence on membrane lipid peroxidation, ROS accumulation, and key enzyme activities and non-enzyme antioxidants in the AsA-GSH cycle of soybean leaves. Spraying S3307 could improve key enzyme activity at some extent to promote oxidation ability, so as to reduce the harm caused by waterlogging stress.

Regulation Effects of Photoperiod on Growth and Leaf Endogenous Hormones in Broomcorn Millet

WANG JunJie,TIAN Xiang,QIN HuiBin,WANG HaiGang,CAO XiaoNing,CHEN Ling,LIU SiChen,QIAO ZhiJun

Scientia Agricultura Sinica. 2021, 54(2): 286-295. doi:10.3864/j.issn.0578-1752.2021.02.005

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【Objective】In this study, the variation law of photoperiod on physiology and ecology index of broomcorn millet were observed, which would facilitate to develop the genetic regulation network and map related genes of photoperiod.【Method】The experiments were conducted using 3 broomcorn millet varieties (N1-light insensitive-Neimi 1, N2-light middle-Neimi 2, and N3-light sensitive-Ningmi 14) with different light insensitive degree and with four photoperiod treatments in the pot. The phenological period was investigated. The endogenous hormone content at heading stage, plant height and grain protein content at mature period, and net photosynthetic rate of light insensitive variety after heading were measured.【Result】The growth period and plant height of broomcorn millet were significantly increase by long day compared with short day, which in light sensitive material N3 was 62.07% and 104.24% higher than that of short day, and light insensitive material N1 was 3.51% and 33.35%, respectively. The net photosynthetic rate of leaves after heading showed a significant difference with the development of the growth process, the net photosynthetic rate increased significantly with the prolonged light for accumulating more dry matter. Different photosensitive materials had different regulating mechanisms of endogenous hormones under different photoperiod treatments. The accumulation of IAA content to a certain concentration could promote the plant heading. IAA was significantly positively correlated with GA and ZR, with correlation coefficients of 0.51 and 0.40, respectively, while it was significantly negatively correlated with ABA, with the value of -0.62. The ABA content was negatively correlated with GA and ZR, with the value of -0.70 and -0.39, respectively, and the GA content was positively correlated with ZR, with the values of 0.47. The grain protein content of different light-sensitive materials under the short day in the whole growth period was significantly higher than that of other treatments. The grain protein content of N1, N2 and N3 under short day in the whole growth period were 18.89%, 80.50% and 61.23% higher than those of long day in the whole growth period, respectively, indicating that the light-sensitive material was more susceptible to short-day sunshine than the light-insensitive material.【Conclusion】The photoperiod significantly affected the growth and endogenous hormone content in different photosensitivity materials. Long day significantly prolonged the growth period, increased plant height and enhanced leaf net photosynthetic rate, but decreased the grain protein content in broomcorn millet. IAA content to a certain concentration could promote the transformation of broomcorn millet from vegetative to reproductive growth.

Characterization of Fungal Community Structure in the Rhizosphere Soil of Healthy and Diseased-Verticillium Wilt Potato Plants and Carbon Source Utilization

ZHAO WeiSong,GUO QingGang,SU ZhenHe,WANG PeiPei,DONG LiHong,HU Qing,LU XiuYun,ZHANG XiaoYun,LI SheZeng,MA Ping

Scientia Agricultura Sinica. 2021, 54(2): 296-309. doi:10.3864/j.issn.0578-1752.2021.02.006

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【Objective】The objective of this study is to research the differences of the structure and functional diversity of fungal community in the rhizosphere soil of healthy and diseased potato plants, and to clarify the relationship between the structure of soil fungal community and the occurrence of verticillium wilt, so as to provide a theoretical basis for the final explanation of the causes of potato verticillium wilt and ecological control from the perspective of microbial ecology.【Method】The rhizosphere soil of healthy and diseased plants of verticillium wilt of potato was collected from Bashang area in Hebei Province. The number of ITS gene copies of Verticillium dahliae was determined by real-time quantitative PCR, and the structure of soil fungal communities was measured by high-throughput sequencing (Illumina MiSeq). The quantity of V. dahliae and the community of soil fungi were analyzed. The redundancy analysis (RDA) was used to determine the correlation between fungal community structure and soil nutrient. Meanwhile, the utilization capacity of rhizosphere soil microorganisms of healthy and diseased plants to carbon source was compared by Biolog-Eco plate method.【Result】The occurrence of verticillium wilt of potato was related to the ITS gene copy number of V. dahliae in soil. The ITS gene copy number of pathogens was high in rhizosphere soil of diseased plants, but not detected in healthy plants. Illumina MiSeq analysis showed that the diversity index of fungi in rhizosphere soil of diseased plants was lower than that of healthy plants, but the diversity difference was not significant. Compared with the rhizosphere soil of healthy plants, at phylum level, the relative abundance of Ascomycota and Mortierellomycota was increased by 20.68% and 16.16%, respectively, while that of Basidiomycota was decreased by 51.43%. At genus level, the average relative abundance of Verticillium, Penicillium, Vishniacozyma, Rhodotorula and Cladosporium in rhizosphere soil of diseased plants was increased by 71.96, 3.62, 6.11, 15.38 and 6.24 times, respectively, while that of Plectosaphaerella, Guehomyces, Stachybotrys, Gibberella, Aspergillus was decreased by 45.10%, 61.41%, 96.87%, 45.85% and 44.39%, respectively. RDA of fungal community composition and soil nutrient showed that the relative abundance of dominant communities in rhizosphere soil of healthy plants (such as Plectosaphaerella, Guehomyces, Grapevine, Gibberella and Aspergillus) was positively correlated with nitrate nitrogen (NO₃ ^--N), organic matter (OM) and pH, while the relative abundance of dominant communities in rhizosphere soil of diseased plants (such as Verticillium, Alternaria, Colletotrichum, Mortierella, Humicola, Penicillium, Vishniacozyma, Rhodotorula and Cladosporium) was positively correlated with inorganic phosphorus (IP) and available phosphorus (AP). The AWCD values of rhizosphere soil of healthy and diseased plants indicated that the utilization ability of microorganisms to carbon source in rhizosphere soil of diseased plants was higher than that of healthy plants. Further analysis showed that, compared with the healthy plants, the utilization ability of rhizosphere soil microorganisms to carboxylic acids carbon source was significantly improved, while the utilization ability to amino acids, amines, carbohydrates, polymers and miscellaneous was not significantly different.【Conclusion】The decrease of fungal diversity and the change of community structure in rhizosphere soil of healthy and diseased plants are the important characteristics of potato verticillium wilt. The significant increase of relative abundance of Verticillium is the most important characteristic, and the structure of fungal community is affected by soil nutrient. At the same time, the utilization of carboxylic acids carbon source is significantly increased by rhizosphere soil microorganisms of diseased plants.

Expression of Stilbene Synthase Genes from Chinese Wild Vitis quinquangularis and Its Effect on Resistance of Grape to Powdery Mildew

DING Xi,ZHAO KaiXi,WANG YueJin

Scientia Agricultura Sinica. 2021, 54(2): 310-323. doi:10.3864/j.issn.0578-1752.2021.02.007

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【Objective】Two stilbene synthase (STS) genes from Chinese wild Vitis quinquangularis ‘Danfeng-2’ (VqSTS9 and VqSTS21) were cloned and functionally investigated with the aim to provide a theoretical basis for improving the disease-resistance and quality of V. vinifera.【Method】Homologous cloning was applied to obtain VqSTS9 and VqSTS21 and their overexpression vectors under the control of CaMV35 promoter were constructed, respectively. The calluses were induced from single bud segment of V. vinifera cv. ‘Thompson Seedless’, which were used as the receptor materials. The resistant ‘Thompson Seedless’ plants were obtained via Agrobacterium-mediated genetic transformation. Furthermore, the disease-resistant transgenic plants were determined by different levels of detection. The leaves of wild-type (WT) and transgenic plants were inoculated with Uncinula necator, the pathogenic conditions of leaves infected with U. necator were observed by microscope to compare their resistance. The expressions of STS and other genes in the metabolic pathway of resveratrol synthesis were analyzed by quantitative real-time PCR (qRT-PCR) in WT and transgenic plants under natural conditions and after inoculation with U. necator. The types and contents of stilbenoids in transgenic plants were determined by HPLC.【Result】The cDNA sequences of VqSTS9 (JQ868689) and VqSTS21 (JQ868677) from the Chinese wild V. quinquangular accession ‘Danfeng-2’ were cloned, and both were 1 179 bp in length. Four VqSTS9-overexpressing and three VqSTS21-overexpressing plants were confirmed by PCR and Western blot analysis. These transgenic plants enhanced resistance to powdery mildew and reduced hyphae growth compared with WT plants through observing the growth of U. necator. The qRT-PCR results indicated that VqSTS9 and VqSTS21 transgenic plants increased the expressions of STS, its upstream PAL, the downstream RSGT, and transcription factor genes MYB14, MYB15 under the natural conditions when compared with WT plants. However, the expression of CHS was down-regulated. After inoculation, the expression of STS in transgenic plants was significantly up-regulated compared to WT. HPLC determination demonstrated that the stilbenoids mainly existed as the form of trans-piceid, and its content in transgenic plants was higher than that in WT under natural conditions. After inoculation, the expression of STS was induced, and trans-piceid, trans-resveratrol and viniferin were also produced. Compared with WT plants, the types and contents of stilbenoids in transgenic plants increased.【Conclusion】Overexpression of VqSTS9 and VqSTS21 in ‘Thompson Seedless’ can enhance the expression of STS, increase the types and contents of stilbenoids, and further inhibit the growth of U. necator. Therefore, ‘Danfeng-2’ is an important germplasm resource for disease-resistant breeding and quality improvement of V. vinifera. Stilbene synthase genes VqSTS9 and VqSTS21 can improve resistance to powdery mildew of V. vinifera.

Effects of Four Insecticides LC₂₅ on Feeding Behavior of Q-Type Bemisia tabaci Adults

HE YunChuan,WANG XinPu,HONG Bo,ZHANG TingTing,ZHOU XueFei,JIA YanXia

Scientia Agricultura Sinica. 2021, 54(2): 324-333. doi:10.3864/j.issn.0578-1752.2021.02.008

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【Objective】The objective of this study is to investigate the effects of sub-lethal concentration imidacloprid, pymetrozine, spirotetramat, and cyantraniliprole on the feeding behavior of Q-type Bemisia tabaci adults on tomato seedlings, the feeding behaviors of insects were recorded by the thorn-suck potential technique electronic penetration graph (EPG) after treating with four insecticides LC₂₅ for 24 h.【Method】The Q-type B. tabaci adults that were not exposed to pesticides were used as test insects, and Fenyin No.3, the main tomato variety was employed as the test plant. These plants fed by whitefly were well managed in the lab with hydroponic condition for the experiment. The sub-lethal concentration LC₂₅ of four systemic insecticides was determined by bioassay. On this basis, 13 non-phloem and 8 phloem parameters of B. tabaci adults feeding host were monitored by EPG. Moreover, the leaf piercing suction holes were observed with microscopy. No pesticide treatment was set as control (CK).【Result】The rank of LC₂₅values of whitefly adult to four insecticides was cyantraniliprole>pymetrozine>imidacloprid>spirotetramat. Within 8 h monitoring of whitefly on plants, the six kinds of waveforms, including non-probing wave (np), pathway phase wave (C), potential drop wave (pd), phloem activities (waveform E: E1 and E2), and xylem activities wave (G), were occurred after the treatment of LC₂₅of each pesticide. The number of np wave in the cyantraniliprole treatment was largest in all treatments, followed by the spirotetramat treatment. There was no significant difference between the two above pesticides (P>0.05), but was significantly higher than that in CK and imidacloprid treatments (P<0.05). The total duration of C wave and G wave was longest in CK treatment and shortest in cyantraniliprole treatment, and there was significant difference between the two treatments (P<0.05). The total duration of pd wave was longest in CK treatment and shortest in spirotetramat, there was significant difference between the two treatments (P<0.05). The total duration of F wave was longest in spirotetramat and shortest in imidacloprid, and there was significant difference between the two treatments (P<0.05). The total duration of E1 wave and E2 wave was 20.52 and 50.60 min on the phloem of the CK treatment, respectively. There was significant difference with treatments of imidacloprid and cyantraniliprole in the two indicators (P<0.05). However, there was no significant difference with pymetrozine and spirotetramat treatments in the total duration of E1 wave indicator (P>0.05).【Conclusion】The refusal effects on the feeding behavior of Q-type B. tabaci adults have been found by four insecticides with LC₂₅treatments. It provides a theoretical basis for the feeding refusal mechanism study of the sub-lethal effects of B. tabaci and piercing-sucking mouthparts pests on the four pesticides and pest control.

Long-Term Conservation Tillage Enhanced Organic Carbon and Nitrogen Contents of Particulate Organic Matter in Soil Aggregates

LI Jing,WU HuiJun,WU XuePing,WANG BiSheng,YAO YuQing,LÜ JunJie

Scientia Agricultura Sinica. 2021, 54(2): 334-344. doi:10.3864/j.issn.0578-1752.2021.02.009

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【Objective】 Tillage management has essential effects on soil organic carbon (SOC) and total nitrogen (TN). Based on a combined soil aggregate size, particle density and size fractionation method, the study aimed at revealing effects of different tillage treatments on SOC and TN of physical fractions within different sizes of aggregates. This study provided theoretical basis for understanding mechanisms soil carbon and nitrogen sequestration and selecting optimized tillage management for loess hilly region of China. 【Method】 The long-term tillage experiment, started in 1999, was used for the study. The tillage treatments included: reduced tillage without mulch (RT), no-tillage (NT), sub-soiling with mulch (SM), and conventional tillage (CT). The soil samples from the 0-10 cm layer were collected to obtain physical fractions, including free light fraction (LF), coarse and fine particulate organic matter (cPOM and fPOM) and mineral associated organic matter (m-SOM) within four dry-sieving aggregate sizes (>2 mm, 1-2 mm, 0.25-1 mm and <0.25 mm), by applying a combined soil aggregate size, and particle density and size fractionation procedure.【Result】 (1) 15 years application of conservation tillage (including NT and SM) significantly increased SOC and TN contents in 0-10 cm layer. Compared to CT, NT and SM increased SOC content by 22.9% and 21.8%, and increased TN content by 35.2% and 42.3%, respectively. However, RT had no significant effects on SOC and TN contents. (2) Different tillage practices changed the mass distribution, SOC and TN contents of aggregates. Compared to CT, NT and SM improved the mass percentage of 1-2 mm and 0.25-1 mm size aggregates, relatively, increased the mass percentage of >2 mm and<0.25 mm aggregates. Moreover, conservation tillage enhanced SOC and TN contents across four sizes aggregates. Compared to CT, NT and SM averagely increased SOC content in aggregates by 8.5% and 9.5% and increased TN contents by 12.2% and 24.1%, respectively. The fPOM and m-SOM stored largest parts of aggregate SOC and TN, constituting 27.3%-45.1% and 25.0%-52.6% of aggregate SOC, 23.5%-34.7% and 42.2%-64.3% of aggregate TN. Different physical fractions had different reflects to tillage managements. The cPOM and fPOM were the most sensitive fractions. Compared to CT, NT and SM led to higher contents of SOC and TN accumulated in cPOM and fPOM in all aggregates, especially cPOM in >2 mm aggregates and fPOM in <2 mm aggregates.【Conclusion】The long-term conservation tillage (included no-tillage and sub-soiling and mulch management) promoted SOC and TN sequestration in aggregates by increasing the storage of physical protected particulate organic matter in aggregates. Thus, the conservation tillage was a sustainable soil carbon and nitrogen enhancement management for dryland soils for the loess hilly region of China.

Effects of Plastic Film Mulching and Fertilization on the Sequestration of Carbon and Nitrogen from Straw in Soil

WANG ShuYing,LI XiaoHong,CHENG Na,FU ShiFeng,LI ShuangYi,SUN LiangJie,AN TingTing,WANG JingKuan

Scientia Agricultura Sinica. 2021, 54(2): 345-356. doi:10.3864/j.issn.0578-1752.2021.02.010

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【Objective】Crop straws not only contain high content of organic carbon (C), but also are rich in mineral nutrients. Straw returning to field is an important technique for improvement of soil fertility and sustainable development of agriculture in the region of Black Soil in Northeast China. However, the sequestration and characteristics of C and nitrogen (N) from straw in soil under different plastic film mulching and fertilization treatments were not clear. In this study, the contributions of straw C to soil organic C (SOC) and straw N to soil total nitrogen (TN) were quantified to compare the differences of straw C and N in soil among different mulching and fertilization treatments, so as to provide a basis for improvement of soil fertility and protection of Black Soil in Northeast China.【Method】Based on a long-term mulching and fertilization experiment, the ¹³C¹⁵N double-labeled straw was added to the topsoil (0-20 cm) from the different fertilization treatments, including no fertilization (CK), chemical N fertilizer application (N₄), and organic manure combined with chemical N fertilizer (M₂N₂), with/without mulching, and then which were incubated in-situ in the field for 150 days. The contents of SOC and TN and the values of δ¹³C and δ ¹⁵N were measured to analyze the dynamics changes of SOC derived from straw C (¹³C-SOC), TN derived from straw N (¹⁵N-TN) and their ratio with time.【Result】Fertilization, mulching and their interactions significantly influenced the contents of ¹³C-SOC and ¹⁵N-TN (P<0.05). During the whole incubation period, the contribution percentage of¹³C-SOC to SOC (¹³C-SOC/SOC) and that of ¹⁵N-TN to TN (¹⁵N-TN/TN) were 10.48% and 3.18% under M₂N₂ treatment, respectively; the¹³C-SOC/SOC and residual percentage of straw C in soil under fertilization (N₄and M₂N₂) treatments were on average 12.65% and 37.14% under mulching, and averaged 12.08% and 34.50% under no mulching, respectively. On the 150^th day of incubation under the same cultivation mode, the ¹³C-SOC/SOC and residual percentage of straw C in soil were on average 14.33% and 39.40% under N₄treatment and averaged 11.77% and 33.21% in the other fertilization treatments, respectively;¹⁵N-TN/TN under CK treatment was with an average of 4.56%, and was 26.00% and 44.53% higher than that in N₄ and M₂N₂ treatments. The residual percentage of straw N was the highest under CK treatment with/without mulching, with an average of 10.03%, which was the lowest under N₄ treatment without mulching, with a value of 7.87% on the 150^th day of incubation. Regardless of mulching or not, the ratio of ¹³C-SOC to ¹⁵N-TN ranged from 32 to 39 in N₄ treatment, but was lower than 30 in the other fertilization treatments. 【Conclusion】The sequestrations of straw C and N in soil were sensitive to mulching and fertilization. The single application of chemical N fertilizer promoted the accumulation of straw C and the renewal of organic C in soil, and the long-term no fertilization played a positive feedback effect on the sequestration of straw N in soil N pool, while the renewal of soil organic C and N in organic manure combined with chemical N fertilizer lagged behind that in the other fertilization treatments.

Function Analysis of GA₃ Mediate miR171s and Its Target Genes VvSCLs in Grape Seed Development

WANG WenRan,XIE ZhenQiang,ZHUGE YaXian,BAI YunHe,GUAN Le,WU WeiMin,ZHANG PeiAn,ZHENG Ting,FANG JingGui,WANG Chen

Scientia Agricultura Sinica. 2021, 54(2): 357-369. doi:10.3864/j.issn.0578-1752.2021.02.011

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【Objective】The main objective of the present research was to identify the vvi-miR171s and their target genes VvSCL6/15/22 from grapevine genome, and to confirm the role of vvi-miR171s and VvSCL6/15/22 in grape seed development process and their expression pattern in response to exogenous GA₃. 【Method】The mature sequences of vvi-miR171a/b/e/f/g/h were validated by miR-RACE technique from grape (Vitis vinifera L.) cv ‘Wink’. The potential target genes of vvi-miR171s were predicted by PsRNATarget software, and the chromosome localization, phylogenetic, gene structure, conserved domain and motifs analysis were performed by bioinformatics tools. The potential functions of vvi-miR171s and their target genes were predicted by promoters cis-elements analysis. RLM-RACE and PPM-RACE verified the cleavage roles of three target genes by vvi-miR171a/b/e/f/g/h, and the qRT-PCR method was used to detect their temporal expression patterns in grape seed and seedless grape induced by exogenous GA₃. 【Result】Six vvi-miR171s mature sequences in grape were determined and cloned, which were consistent of the sequences of miRBase. Based on this result, the target genes of them, including VvSCL6, VvSCL15 and VvSCL22, were prophesied. The target genes bioinformatics analysis showed evolutionary genetic distance between VvSCL6/22 genes and homologous genes in apple (Monodelphis domestica), peach (Prunus persica), and cherry (Prunus avium) is relatively closed, while VvSCL15 had the same genetic distance from the homologous genes in Arabidopsis, apple, peach and cherry. Meanwhile, the gene structure of VvSCLs was similar to their close genes in phylogenetic tree. All SCL protein sequences had GRAS domain, both element types, arrangement order of motifs. And 5 same motifs for GRAS domain were identified, indicating that the SCL protein structure was relatively conservative, with the conserved functionality. Subsequently, an array of gibberellin and Salicylic acid responsive cis-acting elements were identified in vvi-miR171s and target genes VvSCLs promoter sequences, indicating their possible involvement in the regulation of GA-responsive and SA-responsive grape growth and development. Moreover, the qRT-PCR analysis revealed that the expression of vvi-miR171a was significantly repressed by GA₃ in seed, while its target gene VvSCLs showed reciprocal expression pattern, particularly. There was a strong negative correlation between vvi-miR171a -VvSCL15 in the grape seed of CK and seed region of GA₃treatment. Taken together, vvi-miR171a might the major member of vvi-miR171s in response to GA₃, additionally, it might involve in the grape berry seed development through negative regulating of VvSCL15 gene developmental in response to GA₃. 【Conclusion】vvi-miR171a/b/e/f/g/h were identified from grapevine ‘Wink’, and all of members could cleave three target genes VvSCL6/15/22. The vvi-miR171a-VvSCL15 might be the major member in response to gibberellin and involve in regulating grape seed development.

Identification of F₁ Hybrids in Blueberry (Vaccinium corymbosum L.) Based on Specific-Locus Amplified Fragment Sequencing (SLAF-seq)

LIU YouChun,LIU WeiSheng,WANG XingDong,SUN Bin,LIU XiuLi,YANG YanMin,WEI Xin,YANG YuChun,ZHANG Duo,LIU Cheng,LI TianZhong

Scientia Agricultura Sinica. 2021, 54(2): 370-378. doi:10.3864/j.issn.0578-1752.2021.02.012

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【Objective】 The aim of this investigation was to develop a method of identifying true F₁ hybrids based on next-generation sequencing data, so as to provide an alternative strategy for fruit breeding, genetic analysis and genetic map construction.【Method】A total of 318 F₁ population derived from reciprocal cross between two blueberry (Vaccinium corymbosum L.) accessions were sequenced by specific-locus amplified fragment sequencing (SLAF-seq). Based on the blueberry reference genome, the rare allelic variation, principal component analysis (PCA) and k-means clustering were used to identify the accidental hybrid in blueberry population. Furthermore, homozygous SNP were used to confirm the data.【Result】A total of 65.89 Gb sequence data was generated by Illumina sequencing, in which the GC content was 38.63% and Q30 value was 95.44%. The average sequencing depth of the two parents and F₁ progenies were 12.86× and 5.41×, respectively. By aligning the sequencing reads onto the reference genome of Vaccinium corymbosum, a total of 73 543 and 114 851 SNPs were called in two reciprocal cross populations, respectively. According to these SNPs with minor allele frequency (MAF) more than 0.05, PCA, and k-means analysis, it was yielded four outlier individuals regarded as accidental hybrid progenies. Moreover, ten outliers were identified after total rare-alleles analysis and the private rare-alleles analysis by using SNP with MAF﹤0.05, including the above four individuals. By verifying the accidental hybrid progenies, homozygous SNP between the two parents was accounted for 34.56% and 38.95% of the total SNP, respectively, which were subjected to genotype the ten outliers and conflicted genotypes, and which were observed in individuals except H194-123 with the two parents, and the accuracy of our developed methods was validated.【Conclusion】For species with a reference genome, it was an effective strategy by using allele frequency (MAF) data set to identify the accidental hybrid progeny based on genetic relationship analysis and unique rare allele variation analysis.

Dynamic Analysis for the Characteristics of Flavor Fingerprints for Brown Rice in Short-Term Storage Under High Temperature Stress

LIU Qiang,LIU JiWei,TIAN Tian,YAN Wei,LIU Bing,ZHAO SiQi,HU QiuHui,DING Chao

Scientia Agricultura Sinica. 2021, 54(2): 379-391. doi:10.3864/j.issn.0578-1752.2021.02.013

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【Objective】 Changes of flavor fingerprinting in brown rice during high temperature short-term storage were investigated to establish a novel non-destructive detection methods for quality prediction of brown rice.【Method】 The brown rice with moisture content of 17.0 % (wet basis) was stored for 0, 5, 10, 15, 20 and 25 days under 40℃ temperature with relative humidity of 70%. Brown rice flavor compounds were collected by using headspace solid phase extraction method and its fingerprinting characteristics determined via gas chromatography-mass spectrometry (GC-MS) and gas chromatography-ion mobility spectrometry (GC-IMS). Dynamic principal component analysis (dPCA) model based on GC-IMS datasets was constructed.【Result】 Total of 42 volatile organic compounds were identified and quantitated based on GC-MS datasets. Aldehydes, alcohols and esters were the major compounds in flavor of fresh brown rice. After high temperature 15 days of storage, the contents of hexanal increased (P < 0.05) to (185.18±15.71) μg·kg^-1 from initial contents of (81.09±0.53) μg·kg^-1, and then sharply decreased to (12.89±0.72) μg·kg^-1 aat 25 days of storage. Volatile organic compounds, including benzyl alcohol, 1-octene-3-ol and styrene, were successfully identified during storage stage. Based on GC-IMS analysis, ethyl acetate could be selected as the potential volatile markers to evaluate the freshness of brown rice. Good discriminated results were obtained for classifying different storage period of brown rice under via dPCA algorithm based on GC-IMS datasets. GC-IMS could better qualitatively analyze the volatile alcohols and ketones compounds than GC-MS. 【Conclusion】 GC-MS and GC-IMS detection method with dPCA discrimination analysis could obtain reliable flavor fingerprinting, achieve outstanding evaluation performance, and provide technical support for qualitative and quantitative monitoring the freshness of brown rice during high temperature storage.

Application of Stable Isotope Technology in the Origin Traceability of Sheep

WANG Qian,LI Zheng,ZHAO ShanShan,QIE MengJie,ZHANG JiuKai,WANG MingLin,GUO Jun,ZHAO Yan

Scientia Agricultura Sinica. 2021, 54(2): 392-399. doi:10.3864/j.issn.0578-1752.2021.02.014

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【Objective】By comparing the differences of carbon and nitrogen stable isotopes in different parts of mutton and analyzing the stable isotopes of mutton, whole bone meal, de-fatted bone meal and bone collagen, the technical support was provided for the origin traceability identification of sheep. 【Method】Isotope Ratio Mass Spectrometer (IRMS) was used to determine δ ¹³C and δ ¹⁵N values in different parts of sheep, and δ ¹³C and δ ¹⁵N values in de-fatted mutton, whole bone meal, de-fatted bone meal and bone collagen of the sheep bones from different origins (New Zealand, and Ningxia, Gansu, Anhui, China). ANOVA test, linear discriminant analysis and correlation analysis were carried out on the results to determine the traceability of carbon and nitrogen stable isotope to the origin of sheep. 【Result】The δ ¹³C values of hind legs and ribs were significantly different from those of chest fork, abdomen and neck (P<0.05), and there was no significant difference in δ¹⁵N values among the five parts of hind legs, chest fork, ribs, abdomen and neck (P>0.05). The δ¹³C and δ ¹⁵N values of de-fatted mutton, whole bone meal, de-fatted bone meal and bone collagen from different origins (Ningxia, Gansu, Anhui and New Zealand) were significantly different (P<0.05). De-fatted mutton had the best effect on the origin traceability, with the original discrimination accuracy rate was 84.9% and the cross-validation discrimination accuracy rate was 82.4%. The accuracy rate of distinguishing the origin by whole bone meal and bone collagen was over 65%. The correlation analysis results show that de-fatted mutton, whole bone meal, de-fatted bone meal and bone collagen had extremely significant correlation (P<0.01), and the carbon isotope in bone collagen had the highest correlation with de-fatted bone meal and whole bone meal, with correlation coefficients of 0.903 and 0.866, respectively. 【Conclusion】Stable isotope could effectively identify sheep samples from different origins, and the change trend of stable isotope composition in mutton, whole bone meal, de-fatted bone meal and bone collagen was basically the same, so it was feasible to trace the origin of sheep by using stable isotope technology.

CRISPR/Cas9 Mediated Exogenous Gene Knock-in at ROSA26 Locus in Sheep Umbilical Cord Mesenchymal Stem Cells

LI SongMei,QIU YuGe,CHEN ShengNan,WANG XiaoMeng,WANG ChunSheng

Scientia Agricultura Sinica. 2021, 54(2): 400-411. doi:10.3864/j.issn.0578-1752.2021.02.015

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【Objective】The use of CRISPR/Cas9 system to establish a sheep tracing umbilical cord mesenchymal stem cell line lays the foundation for the clinical therapy and mechanism of MSCs. 【Method】Three single guide RNAs (sgRNA)were designed and synthesized for the ROSA26 locus of sheep by using the online tool ZiFiT Targeter Version 4.2, PCR was performed using the point mutation method with the px330 plasmid as a template. The PCR product was circularized after the plasmid DNA was removed by Dpn I, the sgRNA / Cas9 vector targeting sROSA26 was constructed after enzyme digestion and sequencing identification. The constructed plasmid contained Cas9 and sgRNA expression cassettes, which were driven by the U6 promoter. Because of the off-target effect of CRISPR/Cas9 system, the vector was transfected into sUMSCs, after extraction of its genome for PCR, T7E1 enzyme digestion. Then analyzing the gray levels of the bands to detect the vector editing activity. The sgRNA / Cas9 vector can cuts the double-stranded DNA at the target site. In order to knock in the reporter gene, the Donor vector needs to repair the sequence on the donor DNA by homologous recombination at the DNA break to insert target sequence. Based on the sgRNA sequence, the left and right homology arm amplification primers were designed and synthesized at the upstream and downstream of the sROSA26 locus. Using sheep's whole genome as a template for PCR amplification to obtain left and right homology arms. After recovery and purification, recombinant with pMD19-Simple to obtain left and right homologous recombinant plasmids. The left and right homology arm plasmids were ligated with the Donor expression vector DC-DON-SH02 ROSA26 to obtain the left arm recombinant targeting vector. The homologous recombination vector carrying green fluorescent protein (GFP) and puromycin resistance gene was constructed. The survival time of cells with different concentrations of puromycin was observed in well-growing sUMSCs to determine the optimal concentration and time of resistance screening. In sUMSCs, sgRNA / Cas9 vector and Donor vector were co-transfected by liposome method. Puromycin resistance screening was performed 48 hours after transfection. After the screening, cells need to be replaced with normal media and continue to expand. Observing the expression of GFP and extracting the positive cell genome , designing two pairs of upstream and downstream primers for ROSA26 site for PCR to detect its editing status. 【Result】(1) Three sgRNA primers were designed for the sheep ROSA26 locus. The expression vector px330-sgRNA1/2/3 were successfully constructed by point mutation method and the vectors were transfected into sheep umbilical cord mesenchymal stem cells. T7E1 enzyme assay results indicated that the editing efficiency of px330-sgRNA1 was the highest, about 20%. (2) Based on sgRNA1, the left and right homology arms of the targeting vector were obtained by PCR, and the recombinant vector(sROSA26-HA) of sheep ROSA26 locus was successfully obtained by a series of molecular biological methods; (3) px330-sgRNA1 and sROSA26-HA were co-transfected into sheep umbilical cord mesenchymal stem cells using Lipofectamine2000, and positive colonies could express green fluorescent protein were obtained after 1.5 μg·mL ^-1 puromycin selection for 15 days. PCR detection of one clone indicated that the targeting vector had been integrated into the genome. 【Conclusion】The exogenous GFP gene could successfully integrated in specific locus of sheep umbilical cord mesenchymal stem cells using the CRISPR/Cas9 system, and these cells can be used to trace the position and differentiated cell type of MSCs. This research laid a foundation for further clinical transformation of mesenchymal stem cells.

Preparation and Expression of rhPA/GH Double Transgenic Rabbits

SONG ShaoZheng,YU KangYing,ZHANG Ting,LU Rui,PAN ShengQiang,CHENG Yong,ZHOU MingMing

Scientia Agricultura Sinica. 2021, 54(2): 412-421. doi:10.3864/j.issn.0578-1752.2021.02.016

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【Objective】The integration of the double genes is able to promote the expression level of the target genes in the transgenic organisms. The aim of this study was to obtain the rhPA/GH double transgenic rabbits, and then the expression level of the target gene rhPA and the individual growth and development of these rabbits were compared and analyzed, so as to provide a new new approach for preparation of high expression rhPA transgenic animals and genetic breeding. 【Method】 Not Ⅰ/ Sal Ⅰ double enzyme digestion PCL25/GH plasmid and QIAGEN DNA gel purification kit were used to recover gene fragments for microinjection. The three rhPA single-transgenic rabbits (K06, K10 and K17) were used as donors. The rhPA/GH double-transgenic rabbits were obtained by FSH/hCG superovulation, pronuclear microinjection of fertilized eggs, embryo transfer, phenol / chloroform extraction of newborn rabbit’ ear tip tissue genome and PCR integrated detection. In addition, the expression levels of rhPA in single-transgenic rabbit and double-transgenic rabbit whey were compared by ELISA and Western blotting. The body weight of transgenic rabbits at different months was measured, and the effects of GH on growth and development of rabbits were analyzed by body weight at growth stage. 【Result】The about 16 700 bp of microinjection gene fragments were successfully obtained. A total of 122 eggs were obtained from 3 donor rabbits, 103 of which were fertilized, and the fertilization rate was 84.4% (103/122). After microinjection, 81 fertilized eggs with good morphology were selected and transplanted into 6 recipient rabbits by synchronous estrus. Five rabbits were pregnant and the pregnancy rate was 83.3% (5/6). A total of 32 offspring were born at the end of pregnancy. There were 19 rhPA transgenic rabbits identified by PCR, and 11 of them were the rhPA/GH double transgenic rabbits (7♂, 4♀), so the double-gene integration rate was 34.4% (11/32). The four rhPA/GH double-transgenic female rabbits were derived from K06 donor rabbits (labelled K06-1 and K06-2), K10 donor rabbit (labeled K10-1) and K17 donor rabbit (labeled K17-1), respectively. The rhPA expression in single transgenic rabbit whey of No. K06 was 42.2 μg·mL ^-1, and the rhPA expression in the double transgenic rabbit whey of No. K06-1 and K06-2 was 432 and 444 μg·mL ^-1, respectively. The rhPA expression in the single transgenic rabbit whey of No. K10 was 42.8μg·mL^-1, and the rhPA expression in the double transgenic rabbit whey of No. K10-1 was 636 μg·mL ^-1. The rhPA expression in single transgenic rabbit whey of No. K17 was 15.2μg·mL^-1, and which in double transgenic rabbit whey of No. K17-1 was 248 μg·mL ^-1. The expression of rhPA in mammary glands of four female rhPA/GH double-transgenic rabbits (K06-1, K06-2, K10-1, and K17-1) were 248-636 μg·mL^-1, which was much higher than that of rhPA single-transgenic rabbits (K06, K10, K17, expression levels: 15.2-42.8μg·mL ^-1). That is to say, the expression level significantly increased about 10.2 to 16.3 times, and it showed that GH could synergistically promote the expression level of target gene rhPA in the mammary glands of transgenic rabbits. Besides, the western blotting results showed a band of about 39.0 kDa, which was the same size as the target protein rhPA, further proving that this protein expressed in the breast of transgenic rabbits was the target product rhPA. By measuring the weight of rhPA/GH double-transgenic rabbits from their birth to 6 months continuously, it was found that the weight was no significant difference between the rhPA/GH transgenic rabbits and the normal non-transgenic rabbits. The growth curve was drawn to further indicate that 4 transgenic rabbits with integrated GH and 2 normal rabbits without integrated GH had no significant difference in body weight at different stages of growth and development. The body weight for 6 months was about 4.0-5.0 kg. This proved that the introduction of GH did not have harmful influence on their survival and normal growth and development to adulthood. 【Conclusion】In this experiment, rhPA/GH double-transgenic rabbits were successfully prepared, and it has been proved that the introduction of GH could significantly increase the expression level of rhPA. Moreover, it could not affect the growth and development of transgenic rabbits. This laid a foundation for the preparation of high expression transgenic rabbits and other animals in the future, and also provided a new idea and method for the establishment of transgenic animal mammary gland bioreactors and transgenic breeding..

Comparison of Growth Physiology and Gut Microbiota Between Healthy and Diarrheic Lambs in Pre- and Post-Weaning Period

YANG NingZhi,LI Ting,WANG Yan,CHEN Zhuo,MA YiCheng,REN QiangLin,LIU JiaJia,YANG HuiGuo,YAO Gang

Scientia Agricultura Sinica. 2021, 54(2): 422-434. doi:10.3864/j.issn.0578-1752.2021.02.017

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【Objective】Lamb diarrhea is one of the common clinical diseases caused by the certain pathogens or the interaction with malnutrition, abnormal management, and environmental stress during newborn and weaning periods, which hazards the lamb production. There is lack of enough investigation about the differences of lambs' physio-biochemistry with their gut microbiota caused by diarrhea between suckling and weaned periods. Therefore, this study was intended to compare the growth physiology with gut microbiota of diarrheic lambs in pre-and post-weaning period, so as to lay a scientific foundation for prevention and treatment of lamb diarrhea occurred at different growth stages precisely and effectively. 【Method】10 suckling healthy lambs and 10 diarrheic lambs at age of 31-45 day, and another 10 weaned healthy lambs and 10 diarrheic lambs at age of 60-75 day were selected and paired to check the growth status, blood physio-biochemistry and inflammatory factors. The rectal fecal samples were used to analyze gut microbiota composition and structure using 16S rRNA. 【Result】1. There was no significant difference in body weight between healthy and diarrhea lambs before weaning, but after weaning, the body weight of diarrheic lambs was extremely lower than that of healthy lambs (P<0.01), and the body temperature, pulse and respiratory rate of diarrheic lambs were also significantly lower than those of healthy lambs (P<0.05). 2. Before weaning, the total protein, globulin, and cholesterol in diarrheic lambs were significantly lower than those in healthy lambs (P<0.05). After weaning, the total number of leukocytes in diarrheic lambs was extremely higher than that of healthy lambs (P<0.01). And the blood urea, nitrogen, creatinine, phosphorus, glucose and total bilirubin in diarrheic lambs were significantly lower than those in healthy lambs (P<0.05). After weaning, IL-4, IL-6 and IL-8 in diarrheic lambs were significantly higher than those in healthy lambs (P<0.05). 3. The percentage of common OTUs in the gut of healthy and diarrheic lambs after weaning was almost as doubled as that before weaning. Alpha diversity of the gut microbiota in diarrheic lambs before weaning was significantly reduced as compared to their healthy counterpart (P<0.05). And Beta diversity of the gut microbiota weighted PCoA in diarrheic lambs before weaning was significantly different from that in the healthy lambs (Weighted UniFrac Adonis and Anosim tests, P<0.05). At phylum level: before weaning, the relative abundance of Fusobacteria was significantly increased (P<0.05), whereas that of Verrucomicrobia and Actinobacteria was significantly decreased in the gut of diarrheic lambs as compared to their healthy counterpart (P<0.05). After weaning, the relative abundance of Lentisphaerae and Actinobacteria was significantly reduced as compared to their healthy counterpart (P<0.05). At genus level: before weaning, the relative abundance of Fusobacterium and Clostridium were significantly increased, while Akkermansia, Desulfovibrio and Butyricimonas were significantly decreased in the gut of diarrheic lambs as compared to their healthy counterpart (P<0.05). After weaning, the relative abundance of Ruminococcus was significantly decreased, while Desulfovibrio significantly was increased in the gut of diarrheic lambs as compared to their healthy counterpart (P<0.05). 【Conclusion】It was concluded that before weaning the content of blood proteins and the blood fat decreased significantly, whereas after weaning, the elevation of white blood cells and the decrease of CREA, UREA and the blood glucose was significant in diarrheic lambs. Before weaning, the more obvious gut microbiota dysbiosis was observed, whereas after weaning more significant gut inflammatory activity was observed in diarrheic lambs. The ration structure and the environment during weaning time could be the crucial factors for shifting aforementioned differences.

Comparison and Potential Functional Analysis of Long Non-Coding RNAs Between Ascosphaera apis Mycelium and Spore

CHEN HuaZhi,WANG Jie,ZHU ZhiWei,JIANG HaiBin,FAN YuanChan,FAN XiaoXue,WAN JieQi,LU JiaXuan,ZHENG YanZhen,FU ZhongMin,XU GuoJun,CHEN DaFu,GUO Rui

Scientia Agricultura Sinica. 2021, 54(2): 435-448. doi:10.3864/j.issn.0578-1752.2021.02.018

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【Background】Ascosphaera apis is a fungal pathogen that exclusively infects honeybee larvae, causing a sharp decrease of the population of adult honeybees and colony population. Long non-coding RNA (lncRNA), a recently discovered non-coding RNA (ncRNA), plays a vital biological role in various activities such as epigenetics, cell cycle and dose compensation.【Objective】This study aimed to clarify the differences of number, type and expression profile of lncRNAs between A. apis mycelium and spore, and investigate the potential role of the common lncRNAs, specific lncRNAs and differentially expressed lncRNAs (DElncRNAs). 【Method】The purified mycelia (AaM) and purified spores (AaS) of A. apis were respectively sequenced using strand specific library-based lncRNA-seq technology. The expression levels of lncRNAs in AaM and AaS were calculated using FPKM (Fragment Per Kilobase of per Million mapped reads) method. Common lncRNAs and specific lncRNAs were filtered out following Venn analysis. DElncRNAs within AaM vs AaS comparison group were screened out following the standard of P≤0.05 and |log₂fold change|≥1. Upstream and downstream genes of common lncRNAs, specific lncRNAs and DElncRNAs were aligned against GO and KEGG databases to obtain function and pathway annotations. The competing endogenous RNA (ceRNA) regulation networks of common lncRNAs, specific lncRNAs and DElncRNAs were constructed following target binding relationships, followed by visualization using Cytoscape software. RT-qPCR was performed to verify the reliability of the sequencing data.【Result】In total, 108 614 646 and 105 675 408 raw reads were gained from AaM and AaS, and after strict filtering, 107 780 032 and 104 621 402 clean reads were obtained, respectively, with Q20 of 98.76% and 98.72%, and Q30 of 95.84% and 95.78%. A total of 850 lncRNAs were identified. Seven hundred and one lncRNAs were shared by AaM and AaS, and there were 39 and 110 specific lncRNAs. Via cis function, these shared lncRNAs could regulate 3 992 upstream and downstream genes involving in 42 functional terms such as cellular process, metabolism process and catalytic activity; and 117 pathways such as metabolism pathway, biosynthesis of secondary metabolites and biosynthesis of antibiotics. Specific lncRNAs for AaM and AaS could respectively regulate 243 and 672 upstream and downstream genes. In AaM vs AaS comparison group, 255 DElncRNAs were identified and found to regulate 1 479 upstream and downstream genes, which were associated with 41 functional terms including cellular process, metabolism process and catalytic activity; and 107 pathways including metabolism pathway, biosynthesis of secondary metabolites and biosynthesis of antibiotics. Forty one, five and 13 miRNA precursors were predicted from common lncRNAs, specific lncRNAs and DElncRNAs. The result of regulatory network analysis showed the formation of ceRNA networks of mycelium lncRNAs and spore lncRNAs; ten lncRNAs in mycelium could bind to eight miRNAs, further targeting 77 mRNAs; while eight lncRNAs in spore could link to seven miRNAs, further targeting 87 mRNAs; two DElncRNAs including TCONS_00008630 and TCONS_00009302 could simultaneously target miR-4968-y, further regulating ten mRNAs. The result of RT-qPCR suggested the differential expression trend of four DElncRNAs were in accordance of that in sequencing result, indicating the reliability of our sequencing data.【Conclusion】Common lncRNAs, specific lncRNAs and DElncRNAs are likely to affect material and energy metabolisms, autophagy, transcription, MAPK signaling pathway, ubiquitin-mediated proteolysis, proteasome and biosynthesis of secondary metabolites, by regulating the expression of upstream and downstream genes, serving as miRNA precursors or ceRNAs, thus regulating the growth, development, reproduction and pathogenicity of A. apis.

Effects of Different Soybean Varieties on the Yield and Quality of Yuba

ZENG ShiXiao,NIAN Hai,CHENG YanBo,MA QiBin,WANG Liang

Scientia Agricultura Sinica. 2021, 54(2): 449-458. doi:10.3864/j.issn.0578-1752.2021.02.019

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【Objective】Soybean (Glycine max L.) is a crop with high economic value, as it is rich in protein and oil. The current study aimed to explore the correlations between soybean varieties and yuba yield, and the correlations between soybean varieties and yuba in the contents of protein, oil, soluble sugar and isoflavones. This study provides a reference for the production of yuba with high isoflavone. 【Method】24 different soybean varieties from Heilongjiang and Guangdong province were used to make yuba with the same processing method. Then the protein and oil content in soybean and yuba was determined by the Kjeldahl method and the Soxhlet extraction approach, respectively. The soluble sugar contents in soybean and yuba were estimated by the anthrone colorimetric method. Moreover, the isoflavones in soybean and yuba were assessed by high performance liquid chromatography. 【Result】Great difference was observed in the content of protein, oil, soluble sugar, isoflavones and yuba yield among the soybean varieties. The yuba yield from Huaxia 8 was the highest, with production rate of 60.50%, followed by Huachun 2 with production rate of 52.44%, indicating that the two varieties should be ideal varieties for producing yuba. Besides, the productivity of yuba from Suinong 37, Huachun 6 and Heihe 43 were relatively high, reaching 48.59%, 48.37% and 47.91%, respectively. Correlation analysis showed that the yuba yield was positively correlated with the protein content (r=0.598**) and negatively correlated with the soluble sugar content (r=-0.423**) in soybean. The contents of the protein, oil and isoflavones in yuba were positively correlated with those of corresponding traits in the soybean (r=0.700**, r=0.537**, r=0.879**). The soluble sugar content of yuba is positively correlated with the soluble sugar content of soybean (r=0.441*). The protein content in yuba was negatively correlated with the soluble sugar content of soybean (r=-0.519*). The oil content in yuba was significantly and negatively correlated with protein in soybean (r=-0.889**), and was positively correlated with soluble sugar and isoflavone content in soybean (r=0.614**, 0.574**), respectively. The content of isoflavones in yuba was negatively correlated with protein content in soybean (r=-0.589**), and was positively correlated with soluble sugar content in soybean (r=0.568**). 【Conclusion】 The productivity and quality traits of the yuba from soybean varieties were significantly different. Huaxia 8 and Huachun 2 were high-yielding varieties for making yuba. The quality characteristics of yuba were determined by the quality characteristics of soybean varieties, which are mainly determined by the genetic characteristics of soybean varieties.

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