Table of Content

01 April 2018, Volume 51 Issue 7

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

New Resistance Sources of Wheat Stem Rust and Molecular Markers Specific for Relative Chromosomes That the Resistance Genes are Located on

HAN Ran, LI TianYa, GONG WenPing, LI HaoSheng, SONG JianMin, LIU AiFeng, CAO XinYou, CHENG DunGong, ZHAO ZhenDong, LIU Cheng, LIU JianJun

Scientia Agricultura Sinica. 2018, 51(7):  1223-1232.  doi:10.3864/j.issn.0578-1752.2018.07.001

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【Objective】Wheat stem rust, caused by Puccinia graminis Pers. f. sp. tritici Eriks. and E. Henn (Pgt) is one of the most potentially destructive wheat diseases, seriously threatening world wheat production. The emergence of new races Ug99 of stem rust caused the global wheat to be under the threat.Exploring new resistant source of wheat stem rust is one of the effective measures against Ug99. In order to explore stem rust new resistant source, the mixed dominant stem rust physiological races in China were inoculated to 165 wheat-alien species chromosome lines at the seeding stages. 【Method】All of the 165 wheat-alien species chromosome lines, 3 hexaploid wheat and the susceptible control Little Club (LC), were sown in the 10 cm diameter clay pots. When the primary leaves were fully expanded, they were inoculated using talcurediospore powder mixture of the common races 34MKGQM and 21C3CTHSM. The Infection Types (ITs) of the material tested was recorded according to the standard ‘0-4’. Meanwhile, genomic DNA was extracted from stem rust immune, nearly immune, or highly resistant additions/substitutions and their corresponding whole set of chromosome lines and Chinese Spring (CS). PCR was performed on these materials by screening 101 pairs of PLUG primer. PCR products were firstly digested by DNA restricted enzymes TaqI and HaeⅢ, and then were detected through 2.0% agarose gel electrophoresis to screen and establish chromosome-specific molecular marker where the stem rust resistance gene was located on.【Result】Among the 165 wheat-alien species chromosome lines, CS-Ae. geniculata 7M^g#1 addition, CS-Ae. geniculata 7M^g#1(7A) and 7M^g#1 (7B) substitutions, CS-Imperial rye 1R addition, CS-Th. intermedium ?Ai addition (? indicates homoeologous group of the alien chromosomes in wheat background was not identified), CS-Ae. uniaristata 6N addition, CS-Ae. variabilis 6S^vS telosomic addition, CS-Chile barley 6H^ch addition are immune or nearly immune to stem rust. ALCD-Ae. caudata 7C#1 addition, CS-Ae. geniculata 7M^g#1(7D) substitution, CS-Imperial rye 6R addition, CS-Ae. longissima 6S^l#3 addition, CS-Ae. longissima 6S^l#2(6B) substitution, CS-Ae. searsii 3S^s#1 addition, CS-Ae. speltoides 2S#3 addition are highly resistant to stem rust. Comparative analysis of chromosomal locations of stem rust resistant genes indicates that chromosomes 6S^l#2 and 6S^l#3 of Aegilops longissima, chromosome 6R of Imperial rye, chromosome 6H^ch of Chile barley, chromosome 7M^g#1 of Ae. geniculata, chromosome7C of Ae. caudate and ?Ai of Th. intermedium may harbor new stem rust resistance gene (s). Molecular marker screening, localization, specificity verification showed that 8 new molecular markers have been developed. Among them, 5 (TNAC1715, TNAC1718, TNAC1737, TNAC1739 and TNAC1753) and 3 (TNAC1740, TNAC1751, and TNAC1756) have been assigned to chromosomes 6R and 6S^l, respectively. 【Conclusion】 Eight materials which probably contain new gene(s) against stem rust were obtained, and eight new chromosome specific molecular markers for stem rust resistance gene(s) were established.

Fine Mapping of Grain Test Weight Gene tw1 in Maize

SUN CanRan, ZHANG XueHai, MA ZhiHui, GUO ZhanYong, TANG JiHua, FU ZhiYuan

Scientia Agricultura Sinica. 2018, 51(7):  1233-1243.  doi:10.3864/j.issn.0578-1752.2018.07.002

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【Objective】Starch grain density affects maize kernel test weight. In this study, we use a mutant Mrd of maize to identify and fine map the gene controlling starch grain density, which are helpful for the cloning and function verification of the related grain test weight gene.【Method】The Mrd is a starch grain density mutant which was identified during maize breeding practices. Scanning electron microscopy and near infrared spectroscopy (NIR) analyzer were used to observe changes of chemical composition in the Mrd kernels. The segregation population F₂ and BC₁ were derived from the cross of Mrd and B73, which were planted in Zhengzhou and Yuanyang, Henan Province and Sanya, Hainan Province, and used for genetic analysis. BSA (Bulked Segregation Analysis) was used to identify linkage markers selected from 1 000 pairs of SSR primers from maizeGDB (http://www.maizegdb.org) of target gene. The BC₁ segregation population of 38 thousand individuals were used to fine map the target gene tw1. The candidate genes were sequenced and functional predicted by bioinformatics. Allelism test was performed for tw1 and su2. 【Result】Compared with the normal seed, the Mrd had smaller grain size and no change in grain length and increased specific gravity. The crude protein content in mutants decreased, the content of crude starch did not change significantly, but the Mrd has irregular shape and smaller starch grain, increased density, and increased grain test weight. Observations of the starch grain structure inside the kernel in different days after pollination showed that the density of the mutant kernel starch kernel increased with the progress of development and was always higher than that of the normal kernel. The genetic analysis of F₂ and BC₁ population showed that grain test weight mutation was controlled by a single recessive gene tw1 which was firstly located on chromosome 6 between SSR marker umc1105 and bnlg1154. After screening and analysis the recombinant from the BC₁ segregation population, the gene was located between B3 and A47. There are three protein-coding genes in the 0.2 Mb candidate physical interval. Allelism test excluded su2 gene and sequence analysis of the other two candidate genes verified that GRMZM2G042607 should be the primary candidate gene of tw1, which encodes protein with a carbohydrate recognition domain and deposits deposition of carbohydrates in the seed. 【Conclusion】The tw1 gene is fine mapped and find the candidate gene, GRMZM2G042607, encodes beta-1,3 galactosyltransferase.

Correlation Analysis on Transcriptomic and Proteome of Soybean Resistance to Bean Pyralid(Lamprosema indicata)

ZENG WeiYing, SUN ZuDong, LAI ZhenGuang, CAI ZhaoYan, CHEN HuaiZhu, YANG ShouZhen, TANG XiangMin

Scientia Agricultura Sinica. 2018, 51(7):  1244-1260.  doi:10.3864/j.issn.0578-1752.2018.07.003

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【Objective】In order to lay a solid basis for a holistic understanding of the defense molecular mechanism of soybean resistance to Lamprosema indicata in soybean, the correlation analysis of the transcriptomic and proteomic of soybean leaves (the highly resistant line (Gantai-2-2) and the highly susceptible line (Wan 82-178)) under Lamprosema indicate larva feeding for 0 h and 48 h was conducted. 【Method】 In this study, soybeans with high resistance and susceptibility to Lamprosema indicata were selected as the research objects. The gene and protein expression abundance was analyzed for the soybean following the Lamprosema indicata feedings using RNA-Seq and iTRAQ technology, respectively. Then the correlation value between the protein level and the transcription level was calculated.【Result】There were 236 DEPs and 1 064 DEGs, 250 DEPs and 680 DEGs, 213 DEPs and 605 DEGs, 211 DEPs and 468 DEGs identified in HR48/HR0, HS48/HS0, HR0/HS0 and HR48/HS48. The results indicated that the correlations between the quantitative protein and mRNA were 0.156, 0.2687, 0.1149, and 0.035 in HR48/HR0, HS48/HS0, HR0/HS0 and HR48/HS48, and 11, 9, 3 and 4 DEPs/DEGs were identified, of which 11, 9, 2 and 3 with same expression trend, respectively. These differential expression proteins were revealed to be involved in metabolic pathways, ribosome, flavonoid biosynthesis, linoleic acid metabolism, amino sugar and nucleotide sugar metabolism, aminoacyl-RNA biosynthesis, alpha-linoleic acid metabolism, biosynthesis of secondary metabolism, phenylpropanoid biosynthesis, RNA transport, glutathione metabolism, ascorbate andaldarate metabolism. The function correlation analysis results showed that 27 pathways correlated to 101 DEPs and 23 DEGs were identified in HR48/HR0, 15 pathways correlated to 147 DEPs and 16 DEGs were identified in HS48/HS0, 18 pathways correlated to 82 DEPs and 10 DEGs were identified in HR0/HS0, and 1 pathway correlated to 71 DEPs and 2 DEGs were identified in HR48/HS48. Candidate differential expression proteins closely related to insect resistance, such as trypsin inhibitor A-like, kunitz-type trypsin inhibitor, chalcone isomerase 4-like, lipoxygenase-9, alpha-dioxygenase 1-like, linoleate 9S-lipoxygenase 1-like isoform 1, lectin precursor, peroxidase 12-like, stress-induced protein SAM22, scorbate peroxidase 1, cytosolic, and so on. Finally, qRT-PCR analysis confirmed that the two kinds of “omics” results were reliable. 【Conclusion】 In this study, some proteins and metabolic pathways were related to insect-resistant, these differential proteins were directly or indirectly involved in soybean response to Lamprosema indicata.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Relationship Between Temperature, Soil Moisture, and Insecticidal Protein Content in Bt Cotton Boll Shell and the Mechanism of Nitrogen Metabolism

ZHANG Xiang, WANG Jian, PENG Sheng, RUI QiuZhi, LI LiNan, CHEN Yuan, CHEN Yuan, CHEN DeHua

Scientia Agricultura Sinica. 2018, 51(7):  1261-1271.  doi:10.3864/j.issn.0578-1752.2018.07.004

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【Objective】 The effects of different temperature and soil moisture on the insecticidal protein contents in Bacillus thuringiensis (Bt) cotton boll shell were investigated to provide a theoretical reference for the safe and stable utilization of Bt cotton in production.【Method】The study was undertaken on two Bt cotton cultivars Sikang 1 (SK1, a conventional cultivar) and Sikang 3 (SK3, a hybrid cultivar) during 2016 and 2017 growing seasons at Yangzhou University Farm, Yangzhou, China. The study was arranged with two factors that consisted of four temperature (29, 32, 35, and 38℃) and five soil moisture content (40%, 50%, 60%, 70% and 80% field capacity). The potted cotton plants were exposed to the twenty treatments for 4 days. In 2016, the effects of temperature and soil moisture on the insecticidal protein contents in boll shell were determined, and the soluble protein contents, glutamate oxaloacetate transaminase (GOT) activities, protease and peptidase activities were further studied in 2017. 【Result】The highest contents of insecticidal protein for SK1 and SK3 were both observed under the treatment of 32℃/60% field capacity, which was 471.1 ng·g^-1 FW and 351.7 ng·g^-1 FW, respectively. Under the same soil moisture, the insecticidal protein contents for SK1 and SK3 at 32℃ were significantly higher than those for other three temperature conditions. Under the same temperature, the insecticidal protein content under 60% field capacity for SK1 and SK3 was higher. Polynomial regression analysis showed a quadratic relationship between the insecticidal protein contents and the temperature and soil moisture. The binary quadric fitting equations for SK1 and SK3 were: Y=-3230.2+17.2X₁+199.1X₂-0.3X₁²-3.7X₂²-0.7X₁X₂ (r＝0.829^**), Y=-3322.0+40.7X₁+145.2X₂- 0.3X₁²-2.0X₂²-0.3X₁X₂ (r＝0.739^**), respectively, where Y stands for insecticidal protein contents, and X₁ is soil moisture, X₂ is temperature. Based on the equations, the maximum insecticidal protein contents would be obtained under the combination of 31.8℃/57.8% for SK1 and 33.2℃/60.8% for SK3, respectively. The physiological characteristics of nitrogen metabolism showed that the soluble protein contents and the GOT activities were highest in the boll shell for SK1 and SK3 under the combination of 32℃/60% field capacity, whereas the lowest protease and peptidase activities were also detected under this condition. The correlation analysis showed that there was a significant positive correlation between the insecticidal protein contents with soluble protein content and GOT activity (r= 0.613**; r= 0.735**), while the insecticidal protein was negatively correlated with protease activity and peptidase activity (r= -0.724**; r= -0.738**).【Conclusion】 The temperature and soil moisture affected the expression of Bt insecticidal protein by regulating the protein synthesis and degradation. And the relationship between Bt protein contents and temperature and soil moisture could be quantified by a binary quadric fitting equation.

Effects of Intercropping on the Transformation of Carbohydrate Related Substances in Stem of Soybean Seedling Stage and Its Relationship with Leaf Photosynthesis

REN ShengMao, DENG YuChuan, WEN FengJun, Sajad Hussain, PU QuanMing, YU XiaoBo, LIU WeiGuo, YANG WenYu

Scientia Agricultura Sinica. 2018, 51(7):  1272-1282.  doi:10.3864/j.issn.0578-1752.2018.07.005

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【Objective】The purpose of this study was to clarify the mechanism of photosynthetic morphology of soybean at seedling stage in maize/soybean intercropping from the perspective of transformation of related sugars and cellulose synthesis.【Method】 Under soybean monoculture and maize/soybean intercropping systems, the strong shade tolerant soybean (Nandou12) and weak shade tolerant soybean (Nan 032-4) were used as the experimental materials. The photosynthetic rate of leaf and the total carbon, cellulose, soluble sugar, sucrose, and β-1, 3-glucan of stem were measured and analyzed. 【Result】 Compared with soybean monoculture, the leaf photosynthetic rate of intercropping soybean was significantly decreased due to maize shading, but the degree of response was different with different soybean cultivar. In intercropping system, the decreasing degree of strong shade tolerance soybean Nandou 12 was relatively small, and Nandou 12 showed stronger photosynthetic ability than Nan 032-4. The total carbon contents in the leaf and stem of shaded soybean decreased, but the reduction degree of Nandou12 was significantly lower than that of Nan 032-4. Correlation analysis showed that the photosynthetic rate of leaf was highly significantly positive correlation with the total carbon content in the leaf and stem, and the cellulose content in the stem (r=0.952, 0.935, 0.825, respectively, P＜0.01). The result explained that shade of intercropping affected the photosynthesis rate, reduced the accumulation and distribution of photosynthetic products in the stem. Finally, the cellulose content in soybean stem was decrease. But the strong shade tolerance soybean Nandou 12, with the higher photosynthetic rate and accumulation of photosynthetic product, was more suitable planting in intercropping. At seedling stage, the soluble sugar content of intercropping soybean stem was significantly lower than that of monoculture soybean. The contents of β-1, 3-glucan and sucrose in intercropped soybean stem from 30-51 d after germinating were significantly higher than that in monoculture, and in intercropping the carbohydrate conversion rate of two soybean was significantly different. Under the same planting pattern, the content and transformation rate of soluble sugar, sucrose and β-1,3-glucan of soybean stem with strong shade tolerance soybean Nandou 12 were significantly higher than those in Nan 032-4. The analysis of cellulose deposition modes showed that, for the same soybean material, the rapid accumulation time and accumulation rate in monoculture system were higher than that in intercropping system. In the same planting pattern, the cellulose accumulated rate in the stem of Nandou 12 was shorter than that of Nan 032-4, but the difference was small, and the accumulation rate was higher than that in Nan 032-4. It resulted in that the cellulose content in the stem of Nandou12 was significantly higher than that in Nan 032-4.【Conclusion】Shading from maize in intercropping system decreases the photosynthetic capacity of soybean leaves, slow the transportation of photosynthate from leaf to stem, and reduce content of stuffing in stem stuffing. Shading changes the accumulation modes of cellulose in soybean stem, and makes the cellulose content decrease. In intercropping system, the strong shade tolerance soybean Nandou12 can maintain higher photosynthetic capacity and stronger cellulose synthesis ability in the stem, so its’ lodging resistance is strong.

Effects of Irrigation and Nitrogen Coupling on Nitrogen Absorption and Soil Nitrate Content of Winter Oilseed Rape

GU XiaoBo, LI YuanNong, HUANG Peng, DU YaDan, CHEN PengPeng, FANG Heng

Scientia Agricultura Sinica. 2018, 51(7):  1283-1293.  doi:10.3864/j.issn.0578-1752.2018.07.006

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【Objective】 This study aimed at the frequent drought at the stem elongation stage of winter oilseed rape (Brassica napus L.) in northwest China, and local farmers usually applied heavy irrigation and nitrogen (N) fertilizer for high benefits, thus led to serious environmental problems. The objective of present study was to determine the reasonable application amount of irrigation and N fertilizer at stem elongation stage of winter oilseed rape cultivated in northwest China. 【Method】 Ten treatments, including three nitrogen levels (N₀: 0 kg·hm^-2, N₁: 80 kg·hm^-2, N₂: 160 kg·hm^-2) and three irrigation levels (I₀: 0 mm, I₁: 60 mm, I₂: 120 mm) at the beginning of stem elongation stage, and a control (CK) with no irrigation and no nitrogen (no basal nitrogen, no topdressing nitrogen) during the whole growing stage of winter oilseed rape were conducted in this two-year experiments. This study determined the effects of different irrigation and nitrogen levels on aboveground dry matter (ADM), seed yield, oil production, nitrogen uptake, soil nitrate content and nitrogen utilization efficiency of winter oilseed rape. 【Result】 Irrigation or nitrogen application at stem elongation stage significantly improved aboveground dry matter, seed yield, oil production and nitrogen accumulation amount of winter oilseed rape. The depth of soil layer with the peak of soil nitrate was moved down obviously with the increase of irrigation amount, and the peak value was increased with the increase of nitrogen application amount, which showed a clear trend of nitrate leaching with the increase of irrigation and nitrogen amount. Total soil nitrate-N accumulation amount in I₁N₁ was not significantly different with I₀N₀; however, was significantly decreased by 41.9 kg·hm^-2 in comparison to I₂N₂. Soil nitrate-N was mainly distributed in 0-40, 40-80, and 80-160 cm soil layer. Seed yield varied from 1 534 to 3 024 kg·hm^-2 and from 2 318 to 3 746 kg·hm^-2 in 2012-2013 and 2013-2014, respectively. The highest seed yield and oil production were always occurred in I₂N₁, and the lowest were always occurred under CK for both seasons. Compared with I₂N₁, seed yield in I₁N₁ was significantly decreased in 2012-2013, a drought season, while no significant differences were found between I₂N₁ and I₁N₁ in 2013-2014, a wet season. No significant differences were found between I₂N₁ and I₁N₁ in both seasons. Average seed yield and oil production in I₁N₁ were 3 264 and 1 358 kg·hm^-2 for both seasons, respectively, which were just 3.6% and 4.7% lower than I₂N₁, respectively. Nitrogen absorption amount in I₁N₁ was 7.3% significantly lower than in 2012-2013, while there were no marked differences between I₁N₁ and I₂N₁ in 2013-2014. Mean nitrogen agronomic efficiency in I₁N₁ was reduced by 7.2% in comparison with I₂N₁. 【Conclusion】 From the perspective of improving seed yield and nitrogen utilization efficiency of winter oilseed rape, and simultaneously alleviating the downward trend of soil nitrate, I₁N₁ (60 mm irrigation and 80 kg N·hm^-2) treatment can be recommended as a suitable irrigation and nitrogen schedule for winter oilseed rape at stem elongation stage.

PLANT PROTECTION

Development of RT-LAMP Assay for Rapid Detection of Sweet potato feathery mottle virus (SPFMV)

JIANG ShanShan, FENG Jia, ZHANG Mei, WANG ShengJi, XIN ZhiMei, WU Bin, XIN XiangQi

Scientia Agricultura Sinica. 2018, 51(7):  1294-1302.  doi:10.3864/j.issn.0578-1752.2018.07.007

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【Objective】Sweet potato feathery mottle virus (SPFMV) is an important virus infecting sweet potato plants. The objective of this study is to establish a rapid and efficient method for the detection of SPFMV by using reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. 【Method】Four specific RT-LAMP primers for SPFMV detection including SPFMV-FIP (5′-TAAGCGCGGCTGCCTTCATC-CATTCAACCACCCCTGCA-3′), SPFMV-BIP (5′-TCGGTTGTTTGGT TTGGACGGA-ATCAGTTGTCGTGTGCCTC-3′), SPFMV-F3 (5′-GAGTCTTGCGCGATATGCA-3′) and SPFMV-B3 (5′-ACCCC TCATTCCTAAGAGGT-3′) were designed by Primer Explorer V4 according to the nucleotide sequence of SPFMV coat protein (CP) gene in GenBank as well as two specific reverse transcription polymerase chain reaction (RT-PCR) primers for SPFMV detection including SPFMV-F (5′-TCTAATGAGAACACTGAATT-3′) and SPFMV-R (5′-TTGCACACCCCTCATTCCTAAG-3′). Different reaction conditions were optimized in the RT-LAMP in order to improve specificity and sensitivity of the detection, including the primers concentration ratios of F3/B3 to FIP/BIP (1﹕1, 1﹕2, 1﹕4, 1﹕6, 1﹕8 and 1﹕10), dNTPs concentrations (0.025, 0.125, 0.225, 0.325, 0.425, 0.525, 0.625, 0.725 and 0.825 mmol·L^-1), Betaine concentrations (0.4, 0.7, 1.0, 1.3 and 1.6 mol·L^-1), reaction temperatures (59, 61, 63, 65, 67 and 69℃) and reaction times (20, 30, 40, 50, 60, 70, 80 and 90 min). The best reaction conditions were confirmed according to the test results of agarose gel electrophoresis. The RT-LAMP products were identified by sequencing and enzyme analysis. The detection specificity of RT-LAMP was tested by using different RNA templates from SPFMV, Sweet potato virus C (SPVC), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus 2 (SPV2), Sweet potato latent virus (SPLV), Sweet potato virus G (SPVG), Sweet potato chlorotic fleck virus (SPCFV) and leaf sample of healthy sweet potato plant. The sensitivities of RT-LAMP and RT-PCR for detecting SPFMV were compared by using ten-fold serially diluted RNA templates of SPFMV (including original RNA, 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6 and 10^-7 dilutions). Finally, the optimized RT-LAMP and RT-PCR were used to detect the samples of SPFMV collected from Shandong Province. 【Result】The RT-LAMP rapid detection method of SPFMV was established and the optimal amplification was achieved by incubation of 0.8 µmol·L^-1 SPFMV-FIP/SPFMV-BIP, 0.2 µmol·L^-1 SPFMV-F3/SPFMV-B3, 0.325 mmol·L^-1 dNTPs, 1 mol·L^-1 Betaine with template RNA at 65℃ for 70 min. The specificity test showed that the RT-LAMP method established in this study could amplify the typical ladder-like bands only to the RNA carrying SPFMV. The lowest detectable RNA concentration of RT-LAMP was 121.6×10^-4 ng·μL^-1, while the lowest detectable RNA concentration of RT-PCR was 121.6×10^-3 ng·μL^-1, indicating that the sensitivity of RT-LAMP was 10 times higher than RT-PCR for detecting SPFMV. The optimized RT-LAMP method was applied to the detection of SPFMV in field samples and the results were consistent with the visual inspection of RT-LAMV products. It suggested that the RT-LAMP detection method could be applied to detect the SPFMV in the field.【Conclusion】The RT-LAMP established in this study has high sensitivity and speci?city, and is suitable for rapid detection of SPFMV in the field.

Molecular Characteristics and Function Analysis of Cuticle Protein Gene LmNCP1 in Locusta migratoria

YANG YaTing, ZHAO XiaoMing, QIN ZhongYu, LIU WeiMin, MA EnBo, ZHANG JianZhen

Scientia Agricultura Sinica. 2018, 51(7):  1303-1314.  doi:10.3864/j.issn.0578-1752.2018.07.008

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【Objective】The objective of this study is to search and clone a NCP1 from Locusta migratoria (LmNCP1) based on transcriptome database, analyze its sequence and expression characteristics. Then its expression was determined after 20- hydroxyecdysone (20E) induction and interference 20E receptor LmEcR in individuals by RNAi, respectively. Meanwhile, the function of LmNCP1 was analyzed by H&E staining based on RNAi with dsLmNCP1. The results will provide a theoretical basis for the pest control.【Method】A cuticle protein gene was obtained according to the transcriptome database of locusts, and the length of the cDNA sequence was cloned by reverse-transcription PCR (RT-PCR) and sequenced. The gene structure and sequence characteristics were analyzed by using the genome sequence of the locusts. Using the MEGA 6.0 software, the neighbor-joining (NJ) method was used to construct evolutionary tree with the homologous sequences of NCP1 from other insects. Expression profiles of LmNCP1 at different developmental days and in different tissues at day 2 of 5th instar nymphs were assayed using reverse-transcription quantitative PCR (RT-qPCR). Using RT-qPCR, the expression of LmNCP1 was detected after treated with 20E in vivo for 1, 3, 6, 12 h and interfering with 20E receptor gene LmEcR by RNAi for 48 and 72 h, respectively. The biological function of LmNCP1 was analyzed by combination of RNAi and H&E staining method. 【Result】 A cuticle protein gene was obtained by searching the transcriptome database of L. migratoria. The length of the cDNA is 457 bp and full length of its ORF is 306 bp, encoding 101 amino acids. Amino acid sequence analysis showed that the protein encoded by the gene contains one signal peptide and three repeated motifs, but no chitin binding domain, and the repeating motifs were conserved among species by Weblogo analysis. Phylogenetic tree analysis showed that the protein has a close genetic relationship with BcNCP1 of Blaberus craniifer. Thus, the protein was named as LmNCP1 (GenBank accession number: MF326211) according to the result of phylogenetic tree. The results of RT-qPCR showed that LmNCP1 was highly expressed in integument at day 2 of 5th instar nymphs, and lower expression in wing pads, foregut and fat body, but low or no expression in other tested tissues. The expression of LmNCP1 was high at the early stages (N5D1 and N5D2) of 5th instar nymphs, and then decreased (N5D3-N5D6), following a increase at before ecdysis of next instar (N5D7), which is coincidence with the formation of endocuticle and exocuticle. Compared with the control group, the expression of LmNCP1 significantly increased by 1.3 and 0.9 times after injection with 20E for 1 h and 3 h. After 48 h and 72 h of LmEcR interference by RNAi, the expression of LmNCP1 significantly decreased by 71% and 87%, respectively, compared with the control group. After declining the expression of LmNCP1 by RNAi at day 2 of 4th and 5th instar nymphs, the locusts could normally molt and no obvious phenotype was observed. However, the endocuticle of the locusts was thinner than that of the control by H&E staining. 【Conclusion】 A cuticle protein gene LmNCP1 was obtained according to the transcriptome database of L. migratoria. The protein encoded by LmNCP1 does not contain chitin binding domain, but contains three repeated motifs. LmNCP1 was mainly expressed in integument among all the tested tissues. Furthermore, LmNCP1 responds to the regulation of LmEcR-mediated 20E signaling pathway. The results of RNAi suggested that LmNCP1 was involved in the formation of endocuticle during L. migratoria molting.

The cDNA Cloning, Expression Profiling and Functional Characterization of Octopamine Receptor 3 (TcOctβR3) in Tribolium castaneum

LIU XiaoQiang, JIANG HongBo, LI HuiMin, XIONG Ying, WANG JinJun

Scientia Agricultura Sinica. 2018, 51(7):  1315-1324.  doi:10.3864/j.issn.0578-1752.2018.07.009

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【Objective】Octopaminergic signaling system plays a crucial role in the reregulation of behavioral and physiological processes in insects. The red flour beetle (Tribolium castaneum) is a model insect which has been widely used in the study of insect growth, development and physiology. The objective of this study is to utilize T. castaneum as the research insect and elucidate the vital functions of octopamine receptors involved in the physiology and behavior.【Method】Based on the sequence information in GenBank (XP_008198078), the cDNA of an octopamine receptor (TcOctβR3) was cloned by RT-PCR. The open reading frame (ORF), deduced amino acid sequence and the membrane structure domains were predicted by using online service, and phylogenetic tree associated with OctβR3 from other insects was constructed by using neighbor-joining method to clarity its phylogenetic relationship. In addition, the RNA was extracted from different developmental stages (egg, larva, pupa, and adult), different tissues (central nervous system, fat body, midgut, hindgut, malpighian tubule, testis and ovary), as well as the larvae under stress of starvation, respectively. Ribosomal protein S3 gene (TcRPS3) was used as an internal reference. qRT-PCR (real-time quantitative PCR) was employed to determine its expression patterns in different developmental stages, different tissues as well as the induced expression profiles under the stress of starvation. TcOctβR3 was transiently expressed in human embryonic renal cell (HEK293) by using heterologous expression system, and cAMP measuring method was performed to determine the activity of its ability to combine with ligands. Finally, the double stranded RNA of TcOctβR3 was synthesized in vitro, and the physiological functions were verified by track ball behavior analysis as well as RNA interference (RNAi) technology. 【Result】 A complete sequence of TcOctβR3 was cloned with open reading frame (ORF) of 1 305 bp, encoding 434 amino acids, with a signature of 7 transmembrane domains which belongs to the superfamily of G-protein coupled receptors. This gene exhibited a close relationship with the AtOctβR3 from Aethina tumida based on neighbor-joining phylogenetic tree. qRT-PCR results indicated that TcOctβR3 was expressed at all tested developmental stages, particularly high at the early larval stage. However, the expression of TcOctβR3 was not significantly different from other developmental stages. Besides, the expression of TcOctβR3 was remarkably higher in the central nervous system (CNS) than that in other tissues. In the process of starvation for 24 h, the expression of TcOctβR3 significantly fluctuated and then returned to the normal level. The lowest expression of TcOctβR3 was 0.47 fold at 6 h and the highest expression was 1.80 folds at 16 h compared with control, respectively. Based on cyclic AMP response assay, it was found that TcOctβR3 could be activated by octopamine (OA) in a dose-dependent manner with a median effective concentration (EC₅₀) of 8.68×10^-7 mol·L^-1 after heterogenous expression in HEK293 cells. Naphazoline (NA) strongly activated this receptor with EC₅₀ of 8.56×10^-8 mol·L^-1 compared with other ligands. Generally, the rank order for the potency of 4 tested ligands was naphazoline＞tyramine (TA)＞octopamine＞dopamine (DA). RNAi results indicated that the transcript level of TcOctβR3 was significantly knocked down 61.5% by injected the targeted dsRNA. However, no effect on the walking speed and fecundity of T. castaneum adults was observed. 【Conclusion】TcOctβR3 plays an important role in the central nervous system, which modulates the response of the beetle larvae upon starvation. The molecular characterizations were determined based on a cAMP assay, which will provide a solid basis for the future screening of its high efficiency agonists and inhibitors.

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

Acidification Characteristics of Purple Soil, Yellow Soil and Latosol with Electrodialysis Method

CHENG YongYi, LI ZhongYi, BAI YingYan, LIU Li

Scientia Agricultura Sinica. 2018, 51(7):  1325-1333.  doi:10.3864/j.issn.0578-1752.2018.07.010

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【Objective】The soil will be saturated by the ions of H⁺ and Al³⁺ under the process of electrodialysis. Theoretically, the acidification characteristic of soil can be studied with the method of electrodialysis. 【Method】Purple soil, Yellow soil and Latosol were selected to undergo the electrodialysis at a potential gradient of 15 V·cm^-1. The electrodialytic times were 30 times and the duration of each electrodialytic time was 8 hours. The acidification characteristics of three kinds of soil were characterized by the measurements of soil exchangeable acidity, base cations before and after the electrodialysis and the release regulation of base cations in the process of electrodialysis. 【Result】The results showed that the tested soils could be acidified quickly with electrodialysis in short time. The pH value of each soil was less than 4.5, which meant the soils were strongly acidified. After electrodialysis, the contents of exchangeable acidity, exchangeable H⁺ and exchangeable Al³⁺ increased significantly. The contents of exchangeable acidities of Purple soil, Yellow soil and Latosol increased from 3.35, 0.23 and 0.76 cmol(+)·kg^-1 to 18.9, 7.0 and 5.8 cmol(+)·kg^-1，respectively. On the contrary, the content of water soluble and exchangeable base cations, especially Ca²⁺ and Mg²⁺, base exchange capacity and base saturation decreased. For example, the contents of base saturation of Purple soil, Yellow soil and Latosol decreased from 96.8%, 82.6% and 47.3% to 20.5%, 11.8% and 12.2%，respectively. The acidified Purple soil had higher content in soil exchangeable acidity and base saturation. Because the contents of Ca²⁺ and Mg²⁺ were higher than K⁺ and Na⁺ and the electrostatic force between Ca²⁺/Mg²⁺ and negatively charged soil surface is strong. In the process of electrodialysis, the monovalent base cations K⁺ and Na^＋ were released from the soil very quickly. However, the divalent base cations Ca²⁺ and Mg²⁺ were released from the soil slowly and wavily. The exchangeable acidity content of Purple soil was significantly higher than that of Yellow soil and Latosol because of the high content of cation exchange capacity (CEC), which increased the risk of Al³⁺ toxicity of plant in the acidified Purple soil. Meanwhile, the contents of base cations and base saturation of Purple soil were higher than that of Yellow soil and Latosol, because the cations in Purple soil could be supplied from the decomposition of soil minerals. The high content of base cations in the Purple soil ensured the demand of base cation for plant growth and alleviated the further acidification of soil. 【Conclusion】Therefore, the electrodialysis could be as an useful tool to research the acidification characteristics of soil. The acidification characteristic of soil was affected by the content of negative charge on the surface of soil and the soil’s renewed ability of base cations. Compared to Yellow soil and Latosol, the Purple soil had a dual effect on the process of acidification.

Effects of Nitrogen Application on Yield, Water and Nitrogen Use Efficiency of Winter Wheat Under Supplemental Irrigation Based on Measured Soil Moisture Content

JIN XiuKuan, MA MaoTing, ZHAO TongKe, AN ZhiZhuang, JIANG LingLing

Scientia Agricultura Sinica. 2018, 51(7):  1334-1344.  doi:10.3864/j.issn.0578-1752.2018.07.011

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【Objective】Supplemental irrigation based on soil moisture content was a newly water-saving approach developed  in wheat irrigation in recent years, so the purpose of this paper was to evaluate its application potential in Huang-Huai-Hai plain.【Method】In this study, a field experiment of supplemental irrigation based on measurement of moisture content was conducted, combining with different level of nitrogen application. The four irrigation treatments were designed in the main plots, and supplemental irrigation was based on measuring soil moisture content of 0-40 cm soil layers at jointing and anthesis stages of winter wheat. The soil moisture reached 50%FC (field capacity) (W1), 60%FC (W2), 70%FC (W3), 80%FC (W4). In the sub-plots, four nitrogen application treatments were applied at 0 (N₀), 180 (N₁₈₀), 240 (N₂₄₀), 300 (N₃₀₀) kg N·hm^-2. The effects of nitrogen application on yield, water and nitrogen use efficiency in winter wheat under supplemental irrigation based on measured soil moisture content were analyzed.【Result】(1) Under the equal nitrogen application treatments, the wheat yield increased with higher irrigation content. When the supplemental irrigation reached 60%-80%FC range, there were no significant differences in wheat yield among different treatments. For the same irrigation treatments, wheat yield was reduced when the amount of nitrogen applied exceeded 240 kg N·hm^-2. The highest yield (8 104.6 kg·hm^-2) of winter wheat was achieved under the N level of 240 kg·hm^-2 under W₂ treatment in this experimental study. (2) Increasing the amount of nitrogen, supplement irrigation could significantly increase the total water consumption of wheat. However, the effect of nitrogen application was not significant difference when the nitrogen application exceeded to 240 kg N·hm^-2. Supplement irrigation could significantly increase the total water consumption of wheat. When the irrigation reached 60%FC, 70%FC, it significantly reduced the amount of irrigation content compared to the 80%FC, which was beneficial to save irrigation and to achieve higher yields. (3) Under the same nitrogen application treatments, supplement irrigation could significantly increase the water use efficiency of wheat. When the soil moisture reached 60%FC, the highest of water use efficiency was achieved, which was 14.7 kg·hm^-2·mm^-1. For the same irrigation treatments, increasing nitrogen application could significantly improve the water use efficiency of winter wheat, but the amount of nitrogen application should not exceed 240 kg N·hm^-2, otherwise it would decrease the water use efficiency. (4) Under the same nitrogen application treatments, it should control the soil moisture reached 60%FC, and the highest of nitrogen dry matter production efficiency, nitrogen use efficiency were achieved to 60.1 kg·kg^-1, 22.4 kg·kg^-1, respectively. For the same irrigation treatments, the amount of nitrogen applied should be controlled at 240 kg N·hm^-2, and the highest of nitrogen dry matter production efficiency, nitrogen use efficiency were achieved to 63.9 kg·kg^-1, 23.5 kg·kg^-1, respectively.【Conclusion】Under the N level of 240 kg·hm^-2, when the irrigation content at jointing and anthesis stages of winter wheat reached to 60% of soil field capacity, the grain yield, water use efficiency, nitrogen dry matter production efficiency and nitrogen use efficiency were achieved highest. As a result, this mode of nitrogen and irrigation integration was recommended as the regional suitable level for water and fertilizer application.

HORTICULTURE

Cloning and Functional Characterization of an Auxin Response Factor Gene MdARF5 in Apple

AN JianPing, SONG LaiQing, ZHAO LingLing, YOU ChunXiang, WANG XiaoFei, HAO YuJin

Scientia Agricultura Sinica. 2018, 51(7):  1345-1352.  doi:10.3864/j.issn.0578-1752.2018.07.012

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【Objective】The objective of this study is to isolate an apple auxin response factor gene MdARF5, to analyze its expression of exposing to auxin, to identify its role in regulating anthocyanin biosynthesis, then to reveal its biological functions and to provide a theoretical basis for auxin-mediated anthocyanin accumulation. 【Method】 The apple auxin response factor gene MdARF5 was cloned by PCR technology from apple (Malus×domestica ‘Royal Gala’). The phylogenetic tree was constructed by MEGA 5.0 software. The transgenic apple calli were generated via Agrobacterium-mediated transformation. The differences in the anthocyanin accumulation were compared between wild-type and transgenic apple calli. The transient expression assays in tobacco leaves were carried out to test the transcriptional regulation of MdMYB1 gene by MdARF5. 【Result】MdARF5 gene (MDP0000143749) was obtained. The open reading frame (ORF) of MdARF5 contained 2 691 bp, encoding a protein of 896 amino acid residues. Phylogenetic tree analysis showed that the homology of MdARF5 was close to the PbARF5. The transcriptional analysis results indicated that MdARF5 was induced by auxin treatment. On the contrary, the expression levels of anthocyanin biosynthesis genes were repressed. The MdARF5-overexpressing apple calli exhibited decreased anthocyanin content, suggesting that MdARF5 gene might play an important role in regulating anthocyanin accumulation. The sequence of MdMYB1 promoter region was analyzed and a putative ARF binding motif was found. Meanwhile, the transient expression assays were performed in Nicotiana benthamiana leaves and the results showed that MdARF5 could repress the expression of MdMYB1. 【Conclusion】It is speculated that MdARF5 down-regulates anthocyanin accumulation by directly repressing the transcript of MdMYB1.

Effect of RdreB1BI Gene Overexpression on Fruit Quality and Related Gene Expression in Strawberry

YAN YiChao, WAN ChunYan, GU XianBin, GUO ChengBao, CHEN YueHong, GAO ZhiHong

Scientia Agricultura Sinica. 2018, 51(7):  1353-1367.  doi:10.3864/j.issn.0578-1752.2018.07.013

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【Objective】 The effect of exogenous RdreB1BI transformation to the fruit quality and related gene expression in ‘Benihoppe’ strawberry were analyzed to reveal the function and molecular mechanism of quality regulation in transgenic strawberry fruit. 【Method】 The edible organs on five RdreB1BI transgenic strawberry lines and non-transgenic ‘Benihoppe’ strawberries (fruits in full red stage) were used as material to test and determine the vertical diameter, transverse diameter, weight, contents of flavor substance (soluble sugar, soluble protein, ascorbic acid) and coloring materials (anthocyanin, flavonoids and total phenol). The structure of the exogenous gene and seven secondary metabolites synthesis pathway-related genes (RdreB1BI, Fractin, FvC4H, FvCCR2, FvGST, FvF3H, FvDFR and FvMYB306) and the elements of gene promoter were analyzed and were predicted by bioinformatics methods such as BLASTn, GENEFINDER, and PlantCARE. The expression of related genes was detected by qRT-PCR. The data were analyzed by 7300 system software and 2^-△△Ct method. The variance and correlation analysis of physiological, biochemical and molecular data were analyzed. The effects of RdreB1BI on the fruit quality and related gene expression of ‘Benihoppe’ strawberry were discussed comprehensively. 【Result】 The fresh weight of transgenic and non-transgenic strawberries was ranging from 11.75 to 15.42 g. The vertical and transverse diameters were ranging from 35.12 to 40.42 mm and 28.73 to 32.6 mm, respectively. Only the volume diameter of transgenic line 8 was significantly higher than that of line 7, and there were no significant differences between the other samples. The contents of anthocyanin in line 1 and line 7 were significantly higher than those of the control. The contents of total phenol in transgenic line1 and line 7 were significantly higher than that in control. The contents of total phenol in each line were significantly higher than that in the control. The soluble sugar contents in the transgenic line 1, line 7 and line 8 were significantly higher than that of the control (21.70 mg?g^-1 FW), which were 2.87, 3.39 and 3.35 times of the control. There was a positive correlation between soluble solids contents and soluble sugar contents (r=0.811*), but there was no significant difference in the soluble solids contents of the samples. The contents of amino acids in the fruiting fruits of the transgenic lines were ranging from 0.2580 to 0.3950 g/100 g FW. The content of amino acids in the control was 0.5151 g/100 g FW, which was significantly higher than that of the transgenic lines. The contents of titratable acid in the transgenic line 1 and line 7 were significantly higher than that in the control. The content of ascorbic acid in line 1 was 168.35 mg/100 g FW, which was significantly higher than that of the wild-type of 92.50 mg/100 g FW. The contents of soluble protein in transgenic line 9 and line 10 were 25.97 and 25.86 mg?g^-1 FW, respectively, which was significantly higher than that of control (22.93 mg?g^-1 FW). The expression levels of RdreB1BI, FvCCR2 (cinnamoyl-CoA reductase 2-like) and FvMYB306 (myb-related protein 306) in the transgenic lines were significantly higher than that of the wild-type. It was found that the promoter region of differentially expressed genes contained a variety of higher plant cis-acting elements, mainly were CAAT-box and TATA-box, which had enhanced transcription efficiency of eukaryotes. It also included some the cis-acting elements such as G-Box, G-box, MBS, ARE, 5UTR Py-rich stretch, which can affect the response of each gene to light and play a regulatory role in the phenylpropane metabolic pathway. 【Conclusion】 RdreB1BI regulated the related genes involved in the development and maturation of transformants, improved the effectiveness of light, activated the key genes of flavonoid biosynthetic pathway. Differentially expressed genes contained a large quantity of light-induced response elements. The expression of FvCCR2 and FvMYB306 promoted the synthesis of secondary metabolites such as flavonoids, phenols, anthocyanins and total phenols. The transformation of RdreB1BI gene resulted in a significant increase in the nutrient and coloring material contents and improved the fruit quality in strawberry.

FOOD SCIENCE AND ENGINEERING

Optimization of Ultra-High Pressure Sterilization Conditions on the Kiwi Fruit Pulp Produced by Cold Crushing Method and Its Sterilization Effect During Storage Period

YANG TianGe, DENG Hong, LI Han, MENG YongHong, LEI JiaLei, MA Jing, GUO YuRong

Scientia Agricultura Sinica. 2018, 51(7):  1368-1377.  doi:10.3864/j.issn.0578-1752.2018.07.014

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【Objective】 The optimum conditions of ultra-high pressure (UHP) sterilization for kiwi fruit pulp and the effects of sterilization during storage period after ultrahigh pressure treatment were investigated by using Hayward Kiwifruit as raw materials. This study will provide an experimental reference for the non-thermal processing of kiwi fruit and its products development. 【Method】 The kiwi fruit pulp was obtained by cold crushing technology, the response surface method (RSM) was used to establish the mathematical model of ultra-high pressure sterilization conditions for kiwi fruit pulp to obtain the optimum conditions, and the total number of colonies, Vitamin C and browning degree were used as the evaluation indexes. The changes of the total number of colonies, mold and yeast and Escherichia coli group of kiwi fruit pulp were studied with microbiological analysis methods during the storage period at temperatures of 4℃ and -20℃ after ultrahigh pressure treatment. 【Result】 The optimum sterilization conditions of UHP treatment on kiwi fruit pulp were pressure 497 MPa, temperature 27℃, holding time 24 min through the single factor test and the response surface analysis of Box-Behnken model. Under this optimal sterilization condition, the total lethality rates of total number of colonies, Escherichia coli group and mold and yeast were 73.18%, 97.46% and 100.00%, respectively, in kiwi fruit pulp after UHP treatment. Under the standard range, the increments of total number of colonies in kiwi fruit pulp was bigger, which increased significantly of 97.19% and 85.98% respectively after storage for 6 and 14 weeks at 4℃, -20℃, compared with the first day of storage, but the growth rate of total number of colonies was small. While the increments rates of Escherichia coli group, mold and yeast in kiwi fruit pulp after UHP treatment were relatively small, the content of Escherichia coli group, mold and yeast only was 1.36, 0.67 and 0.32, 0.35 lg (CFU/mL) after stored 6 and 14 weeks at 4℃, -20℃, respectively. 【Conclusion】 As a new type of non-thermal sterilization method, ultra-high pressure technology had good sterilization effects for the heat sensitivity materials of kiwi fruit pulp. The kiwi fruit pulp which was used as processing materials and sterilized by the ultra-high pressure technology could be stored 14 weeks at -20℃ still to meet the commercial aseptic requirements, so the ultra-high pressure sterilization combined with the low temperature storage will benefit the further storage, process and utilization of kiwi fruit pulp produced by cold crushing.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Targeted Editing of BMPR-IB Gene in Porcine Fetal Fibroblasts via Lentivirus Mediated CRISPR/Cas9 Technology and Its Effects on Expression of Genes in the BMPs Signaling Pathway

YANG Qiang, XU Pan, JIANG Kai, QIAO ChuanMin, REN Jun, HUANG LuSheng, XING YuYun

Scientia Agricultura Sinica. 2018, 51(7):  1378-1389.  doi:10.3864/j.issn.0578-1752.2018.07.015

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【Objective】 The aims of this study were to edit the BMPR-IB gene in pig fetal fibroblasts (PFF) via a lentivirus-mediated CRISPR/Cas9 genome editing technology, and to investigate its effects on expression of relevant functional genes in the bone morphogenetic proteins (BMPs) signaling pathway. 【Method】 Twenty one single-guide RNAs (sgRNAs) targeting the eighth exon of porcine BMPR-IB gene were designed by the online software http://crispr.mit.edu. The sgRNA sequence with the highest score was selected for annealing with its complementary sequence (including adapters), and then the double-stranded DNA was ligated into the linearized lentiCRISPR v2, with the aim to obtain targeting plasmid. The targeting plasmid was mixed with packing vectors psPAX2 and pCMV-VSV-G at 5﹕4﹕1 molar ratio, then used to produce the recombinant lentivirus in 293T cells. To generate the induction mixture, the lentivirus supernatant was filtered through 0.45 μm, mixed with equal volume of fresh PFF growth medium, and finally polybrene was added to a final concentration of 6 μg·mL^-1. PFF cells were infected in induction mixture and centrifuged at 1 000 g for 1 h at 32℃, then cultured in a 37℃ incubator for 3 days. Three days post-transfection cells were selected with 3.5 μg·mL^-1 puromycin for 6-7 days, and resistant clones targeting BMPR-IB were expanded. Targeting cells were screened first by T7E1 digestion, and then the PCR and PCR-TA cloning were performed to confirm correct targeting. Quantitative real-time PCR was performed to detect the expression levels of relevant functional genes in BMPs signaling pathway. Protein expression of the BMPR-IB gene was detected by Western blotting. Cell Counting Kit-8 (CCK-8) kit was used to measure the proliferation capacity of targeted cells and control group cells. 【Result】 Both T7E1 assay and PCR sequencing showed that the targeted region was successfully edited in targeted cells. TA cloning and sequencing revealed the desired insertion and deletion mutations in the targeted region, and the indels mutation rate was 70%. Moreover, only one off-target site (OTS) was detected among 20 potential ones, and an off-target rate of 10% was observed at this site. Quantitative real-time PCR results demonstrated that the expression levels of BMPR-IB, CylinD2, Cdk2 and Bcl2 genes were significantly (P＜0.01) down-regulated in edited cells compared with wild-type cells. Western blotting results showed that the expression level of BMPR-IB in targeted cell was 38% of that in wild type cells. Cell proliferation assay revealed that the proliferation capacity of targeted cells was significantly lower than that of wild-type PFF cells of the same generation(P＜0.01). Significant losses of proliferation capacity in targeted PFF cells were found following the cell passages (P5, P7 and P9); while there was no significant difference between passage 5 and passage 7 or 9 in control cells. This loss of proliferation capacity in targeted cells does not seem to be caused by the puromycin selection process, as control cells did not show the same loss when underwent the same process. 【Conclusion】 The lentivirus-mediated CRISPR/Cas9 system is efficient for targeted gene editing in PFF. The expression levels of relevant genes in BMPs signaling pathway were significantly down-regulated in BMPR-IB edited cells, indicating that BMPR-IB plays an important role in regulating the proliferation of PFF cells.

Studying the Molecular Mechanism of Heart Development by Using ZBED6 Gene Knockout Pig

WANG DanDan, TANG YuTing, MA YueHui, WANG LiGang, PAN DengKe, JIANG Lin

Scientia Agricultura Sinica. 2018, 51(7):  1390-1400.  doi:10.3864/j.issn.0578-1752.2018.07.016

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【Objective】The ZBED6 gene is a transcription factor that regulates muscle growth and development. In order to investigate the regulatory mechanism of ZBED6 in myocardium growth and development, this study was designed to compare the transcriptome of cardiac tissue obtained from Bama miniature pig between the ZBED6 knockout group (ZBED6-KO) and the wild type group (ZBED6-WT) and to find out the effect of ZBED6 gene knockout on the development and gene expression profile of pig heart tissue. 【Method】 The phenotypic characteristics of ZBED6-KO and ZBED6-WT pig tissues were analyzed by t-test and the expression level of the target gene IGF2 of the ZBED6 gene was quantified by real-time quantitative PCR (q-PCR). Comparing the differences in histological level of tissue structure were performed by paraffin section method. The total RNA was isolated from ZBED6-KO group and ZBED6-WT group. RNA-Seq analysis was performed on Illumina Hiseq 2000 platform. The differentially expressed genes between ZBED6-KO and ZBED6-WT heart tissues were screened by the method of RNA-Seq Pig Sus_scrofa10.2 was used as the reference sequence. Differentially expressed genes were enriched and analyzed with IPA software. 9 differentially expressed genes were randomly selected to verify the reproducibility of RNA-Seq results by q-PCR. 【Result】The heart of ZBED6-KO pig had a larger measured data in weight than ZBED6-WT, which also had a higher expression profile of IGF2 than the latter. Compared with ZBED6-WT pig, ZBED6-KO pig had wide muscle fiber width and less connective tissue. The results suggested that the knockout of ZBED6 gene could promote the growth and development of porcine heart. The sequencing results showed that at least 10 G data were obtained and the clean ratio, Q30 data were more than 90%, of which 62.8% -80.1% of the reads mapped to the pig genome. All of these indicated sequencing was well saturation and the sequencing data was reliable. By analyzing sequencing data, 184 different genes were indicated which contained 114 up-regulated genes and 70 down-regulated genes. Among the 184 genes, there were 141 intact function annotation genes and 43 non-annotated genes. Differential gene hierarchical clustering analysis showed that, the similar expression pattern was detected in ZBED6-KO group (x1, x3, x6), which was also happen to ZBED6-WT group (x2, x4, x5). GO and IPA enrichment analysis obtained 13 significant GO terms, 33 pathways. The difference gene was mainly enriched in the immune response, muscle development, RhoA signal and other related channels. The expression pattern of 9 differentially expressed genes was consistent with the results of RNA-Seq analysis. 【Conclusion】In this study, ZBED6-KO Bama miniature pig was first time to use as the model. We used RNA-Seq technique to explore the effects of ZBED6 gene knockout on cardiac development, which enriched the research of ZBED6 on myocardium development and function.

Identification, Expression, Subcelluar Localization, and Function of glial cell missing (gcm) in Silkworm (Bombyx mori)

ZHANG Kui, PAN GuangZhao, SU JingJing, TAN Juan, XU Man, LI YuTian, CUI HongJuan

Scientia Agricultura Sinica. 2018, 51(7):  1401-1411.  doi:10.3864/j.issn.0578-1752.2018.07.017

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【Objective】The objective of this study is to identify and clone Bombyx mori glial cell missing (Bmgcm), analyze its expression and subcelluar localization, prepare polyclonal antibody and overexpress Bmgcm at the cellular level to explore its function in proliferation and cell cycle, and to provide a theoretical basis for further studying the function of Bmgcm in B. mori.【Method】The full-length of Bmgcm was acquired by RACE technology. Basic sequence and structural information of Bmgcm were analyzed by using several online softwares, including ORF Finder and SMART. Multiple sequence alignment and evolutionary analysis were acquired by Clustalx and MEGA 6.0, respectively. The expression profile was investigated by RT-PCR and real-time PCR. The recombinant protein of Bmgcm was obtained using prokaryotic expression system, and purified with nickel affinity chromatography, then the antibody was prepared. Bmgcm over-expression vector was transfected into B. mori cell line to analyze the subcellular location of Bmgcm, and EDU and flow cytometry were used to study its function. 【Result】 The Bmgcm (BGIBMGA006182) was clustered on nscaf2847 which was located on chromosome 4. The genomic DNA total length of Bmgcm is 4 046 bp, which contains 4 exons and 3 introns. The full-length of cDNA is 1 734 bp, with a 166 bp 5′ UTR, a 227 bp 3′ UTR and a 1 341 bp open reading frame (ORF). This gene encodes 446 amino acid residues, the predicted molecular mass is 50.61 kD and the pI is 5.557, which contains a typical GCM-motif. Multiple sequence alignment demonstrated that GCM motif was highly conserved. Phylogenetic analysis revealed that gcm of insect was clustered alone, and Bmgcm had the highest relationship with gcm from Danaus plexippus. The expression profile revealed that Bmgcm reached peak at the 4th day of embryonic period, and then gradually decreased during embryonic stage. Bmgcm mainly expressed in larval midgut, testis and ovary. The complete ORF sequence of Bmgcm was inserted into prokaryotic expression system, and the recombinant protein of Bmgcm was induced by IPTG and purified using affinity chromatography. Finally, mice were immunized with purified proteins to produce polyclonal anti-gcm antibody. Western blot results indicated that the antibody could specifically recognize the target protein. Moreover, Bmgcm was overexpressed in B. mori cell line, and the result suggested that Bmgcm was located in nucleus. Furthermore, Bmgcm could inhibit cell proliferation and arrested cell cycle in G1/S period. 【Conclusion】 The Bmgcm was identified and cloned, its expression profile and subcelluar localization were analyzed. The available polyclonal antibody was generated by prokaryotic expression, affinity chromatography, and animal immunization. Overexpressed Bmgcm could inhibit cell proliferation and affect cell cycle progression.

AGRICULTURAL ECONOMY & MANAGMENT

Comparison Study on the Level of International Agricultural Modernization Based on the Method of Generation Gap of Industry Elements

HU ZhiQuan, ZHU DianXiao, XIN Ling, HOU LiWei, WANG DongYang

Scientia Agricultura Sinica. 2018, 51(7):  1412-1420.  doi:10.3864/j.issn.0578-1752.2018.07.018

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【Objective】Agriculture modernization is an important part of achieving the ‘four Modernization Synchronization’ development in China. Gap analysis on agricultural modernization development between China and developed countries is helpful to figure out the global positioning of Chinese agricultural modernization. 【Method】 The related references sourced from FAO promoted that agricultural modernization development was a gradual principle process, which was companied with a trend of serious of basic character change. The acknowledgement of the inner principle of agricultural modernization development was the necessary precondition among international comparison. On that base, the paper promoted two core hypothesis, which contained that agricultural modernization development had the common trait, similar historical steps with corresponding characters among different countries; core index of changing rate were same in the steps of agricultural modernization according to different countries, were the base of generation gap to study the comparison of international agricultural modernization development. Therefore, the formulation of generation gap among different index was as follows: D_j=Y_a-W_jb±（X_a-X_jb）/X_jb Based on the related references at home and aboard, a comprehensive index system was built to compare the agricultural modernization development, which was composed of four one-level indexes (Agricultural Economic Benefit, Transformation of Economic Structure, Rural Economic and Social Development and Agricultural sustainable development) and ten second-level indexes. The paper focused on typical countries such as USA, UK, Japan, India, Brazil, South Africa and China to make international comparison, of which, the started early agricultural modernization in the USA, the UK, and Japan could be taken as a long-time comparison in agricultural modernization development due to their historical traits in agricultural modernization development. Meanwhile, Brazil, India and South Africa could be selected as typical countries in comparison, for that the above countries were all belong to developing countries in high speed development, with the many similar opportunities, challenge, policy making and lessons.【Result】The current level of agricultural modernization development in China acted properly as the same level in the end of 1960s to the beginning of 1980s in the USA and the UK, and the beginning of 1990s’ in Japan, which had the similar developing steps in India and Brazil. With respect of sub-index, the unequilibrium agricultural modernization in China was caused by the diversification and low coupling of the core index, which restricted the overall progress of agricultural modernization.【Conclusion】The generation gap analysis was available for evaluate agricultural modernization development, which was easy access to the international comparison and could be used as a tool to analyze agricultural modernization and appraisal agricultural modernization development.