Scientia Agricultura Sinica

Development and Utilization of KASP Marker for Se Concentration in Synthetic Wheat SHW-L1

WEI GuangHui,LI Zhi,CHEN Qiang,LI Yang,CHEN ShiHao,PEI Ying,ZHOU Yong,CHENG MengPing,TANG Hao,WANG JiRui,WEI YuMing,LIU DengCai,CHEN Li,ZHENG YouLiang,PU ZhiEn

Scientia Agricultura Sinica. 2020, 53(20): 4103-4112. doi:10.3864/j.issn.0578-1752.2020.20.001

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【Objective】As one of the essential elements of the human body, selenium plays an important role in disease prevention and antioxidants. Wheat, as one of the main crops, is the main source for humans to obtain plant selenium sources, so increasing the selenium content of wheat grains is an efficient, inexpensive and easy way to supplement selenium. This study intends to develop KASP markers for the identified SNP loci for the breeding of high-selenium wheat.【Method】Using the completed 660K wheat SNP markers of synthetic wheat SHW-L1 recombinant inbred lines population ( recombinant inbred lines, RILs), scanning the RILs population, further narrowing the QTL interval related to the selenium content of the previously located grains, and developing the SNP loci in the stable gene as KASP markers.【Result】Two successfully developed KASP markers AX-1 and AX-2, markers can be used for genotyping and identification of selenium content in the grain of the material. Both markers can be used to classify test materials according to genotype into two categories, namely high-selenium and low-selenium materials, meets the 1:1 allele separation ratio. However, according to the phenotype, the screening rate (greater than 90%) for low-selenium materials is much higher than that of high-selenium materials (less than 30%), and it should be clear that 81% of the phenotype materials with high selenium content have high selenium genotypes, indicating the reliability of the label. Therefore, the KASP markers developed can be used to improve selection efficiency. At the same time, the KASP marker can also be used for genotyping in other derived offspring lines with artificial wheat SHW-L1 as the parent, and the typing results are consistent with the phenotypic results, indicating the validity of the marker.【Conclusion】AX-1 and AX-2 can be used as a practical molecular marker for breeding and germplasm resource creation of high selenium wheat varieties with SHW-L1 as the parent.

Genetic Dissection of Heterosis for Huangzaosi, a Foundation Parental Inbred Line of Maize in China

LI YongXiang,LI ChunHui,YANG JunPin,YANG Hua,CHENG WeiDong,WANG LiMing,LI FengYan,LI HuiYong,WANG YanBo,LI ShuHua,HU GuangHui,LIU Cheng,LI Yu,WANG TianYu

Scientia Agricultura Sinica. 2020, 53(20): 4113-4126. doi:10.3864/j.issn.0578-1752.2020.20.002

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【Objective】The utilization of heterosis is an important approach for high yield breeding in maize. Genetic dissection of heterosis for Huangzaosi (HZS), a foundation parental inbred line of HZS Group, would provide valuable information for its efficient use and high yield maize breeding. 【Method】 A nested association mapping population (NAM), developed from Huangzaosi (HZS) as common parent and 11 diverse inbred lines as other parents and consisting of 2 000 recombinant inbred lines (RILs), were test-crossed with Zheng58 (Z58) and Chang7-2 (CH7), the representative tester in the heterotic pattern of "Improved Reid Group × HZS derived Group" in Chinese maize breeding, respectively. Phenotypic data of two NAM test-cross (NAM-TC) populations were collected at 10 locations across four major corn growing areas of China. Correlations among phenotypic traits and among the NAM and its TC populations, were analyzed. Quantitative trait loci (QTL) mapping of yield and related traits for the NAM and its two TC populations were separately conducted using the model of Joint stepwise regression. Meanwhile, multi-allelic effects of yield related QTL were conducted. Finally, the recombination rates around QTL regions were estimated and compared. 【Result】 Plant height (PH) and yield related traits (mainly refer to kernel number per row, KRPR; kernel weight per hundred, KWPH) simultaneously appeared highly positive correlations with plot yield for both the NAM and its two NAM-TC populations. Compared to the CH7-TC population (with weak heterosis), the lower correlation was observed between the yield performance of the NAM and its Z58-TC population. This meant that the NAM RILs contributed less genetic effects for the yield performance of hybrids in Z58-TC population. A smaller number of QTL were detected for each of the NAM-TC populations. However, the QTL of the NAM-TC populations explained more phenotypic variations than those detected in the NAM per se. Only about 27% of the CH7-TC population QTL and 25% of the Z58-TC population QTL overlapped or neighbored the QTL detected by using the NAM per se. The results of multi-allelic effects of grain yield per ear related QTL showed that HZS contributed about 68.69% of the favorable alleles among those QTL mapped in the Z58-TC population. On the contrary, HZS contributed more adverse alleles among those QTL mapped in the CH7-TC population. A total of 13 yield related genomic regions were identified in the Z58-TC population, whose favorable alleles were mostly contributed by HZS. These genomic regions might play important roles in the formation of yield heterosis of HZS. Moreover, QTL detected in the Z58-TC population tended to locate at the regions with low recombination rate, which was consistent with the feature of heterosis related loci tending to distribute at the genomic regions with low recombination rates. 【Conclusion】 Under the background of Z58, a tester with strong heterosis with HZS, the alleles from HZS greatly contributed to the high yield of hybrids. The QTL detected in the Z58-TC population would tightly associate with the yield heterosis of maize.

Establishment and Application of Multiple PCR Detection System for Glyphosate-Tolerant Gene EPSPS/GAT in Soybean

WEN Jing,GUO Yong,QIU LiJuan

Scientia Agricultura Sinica. 2020, 53(20): 4127-4136. doi:10.3864/j.issn.0578-1752.2020.20.003

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【Objective】This study aims to establish an accurate and efficient high-throughput detection method for herbicide-resistant genes of G2-EPSPS and GAT, providing technical support for the application of transgenic soybean ZH10-6.【Method】Based on the molecular characteristics of glyphosate-resistant soybean ZH10-6 and ZH10, specific primers for endogenous reference genes (Actin), exogenous genes (G2-EPSPS and GAT) and flank sequences (G2EPSPS-2/ZH10P2 and ZH10P1/GAT-2) were designed for testing their specificity and applicability by PCR amplification. The optimal PCR amplified condition of the multiplex PCR system were screened by adjusting primer ratio, DNA template amount, dNTP content, annealing temperature and elongation temperature. The different mix of transgenic soybean ZH10-6 and ZH10 were prepared according to the mass ratio and formed DNA samples of 100%, 50%, 10%, 5%, 1%, 0.5%, 0.1% and 0 for sensitivity detection. Eleven derived lines of transgenic soybean ZH10-6 from different geographical sources were detected for evaluation of its application potential.【Result】Primers of GmActin11 F/R, G2-EPSPS F/R, GAT F/R, ZH10P1/GAT and G2/ZH10P2 were amplified the specific target fragments of 126, 430, 338, 810 and 1 626 bp in the multiple PCR method in transgenic soybean ZH10-6. In addition to the 126 bp target fragment amplified by ZH10 with GmActin11 F/R, the 632 bp target fragments were amplified by ZH10P1/ ZH10P2. The optimal system for multiple PCR amplification included 100 ng DNA template, 5 U·μL^-1 Ex Taq 0.2 μL, 10×ExTaq Buffer 2.5 μL, 2.5 mmol·L^-1 dNTP 2 μL, 10 μmol·L^-1 primers(GmActin11 F/R 0.4 μL, G2-EPSPS F/R 0.6 μL, GAT F/R 0.4 μL, ZH10P1/GAT 0.6 μL and G2/ZH10P2 0.6 μL), ddH₂O supplemented 25 μL. The optimal multiplex PCR amplification procedure: 95℃ for 5 min; 95℃ 30 s, 60℃ 30 s, 68℃ 1 min20 s, 35 cycles; 72℃ 12 min. The sensitivity of the multiplex PCR system is 0.5%, which meets the requirements of the European Union for the labeling of transgenic products. The multiplex PCR method is high specificity and detected ZH10, ZH10-6 and eleven ZH10-6 derived lines successfully. 【Conclusion】The multiple PCR system of EPSPS/GAT were established, which had advantage of high throughput, strong specificity, simple operation and wide application. It can detect transgenic soybean ZH10-6 and its derived lines quickly and accurately.

Effects of Drought Stress During Jointing Stage on Spike Formation and Seed Setting of Main Stem and Tillers of Winter Wheat

LI Ping,SHANG YunQiu,LIN Xiang,LIU ShuaiKang,WANG Sen,HU XinHui,WANG Dong

Scientia Agricultura Sinica. 2020, 53(20): 4137-4151. doi:10.3864/j.issn.0578-1752.2020.20.004

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【Objective】To address the problem of low wheat yield and water efficiency caused by irregular distribution of natural precipitation season and the frequent rate of staged drought in the Huang-Huai-Hai Plain of China, this paper provided theoretical as well as technical provisions for water-saving cultivation of winter wheat in this area by exploring the effects of water insufficiency on the main stem and tiller spikes formation and panicles traits during jointing stage.【Method】A 2-year pot experiment under external rain conditions was carried out from 2017 to 2019 in winter wheat growth season, and the two winter wheat varieties of Shannong 29 and Heng 0628 were used as experimental materials. The total five water treatments were set up during 0-10 d after jointing, including full irrigation treatment during whole growing season as control (CK, maintaining soil relative water content of 75%-80%, the effective soil water content of 42.2-46.7 mm), 0-5 d light drought stress after jointing (T1, maintaining soil relative water content of 65%-70%, soil availability water content of 33.4-37.8 mm), 0-5 d severe drought (T2, maintaining soil relative water content of 45%-50%, available soil water capacity is 15.6-20.1 mm), 0-10 d light drought stress after jointing (T3, maintaining soil relative water content is 65%-70%, soil availability water content of 33.4-37.8 mm), and 0-10 d severe drought stress (T4, maintaining the soil relative water content 45%-50%, availability water content of 15.6-20.1mm).【Result】The results revealed that different degrees of drought stress had no significant effect on the main stem of wheat during 0-10 d after jointing, the effective spike rate of low tiller (Ⅲ, Ⅰp ) decreased rapidly with the increasing the drought level and extension of drought time, while the effective spike rate of high tiller (Ⅱp and Ⅰ1) increased first and then decreased. Light or severe drought at 0-5 d after jointing stage, Ⅱp and Ⅰ1 were higher in effective spike rate, and there was no significant difference in spike number per unit area from CK. Although the effective spike rate of Ⅰ1 and Ⅱp was similar to that of CK in 0-10 d light drought after jointing stage, the percentage of spikes per unit area decreased significantly due to the decrease of Ⅲ, Ⅰp and Ⅳ. The number of spike per unit area of Shannong 29 and Heng 0628 was decreased by 4.94%-5.06% and 6.77%-8.33%, respectively. For 0-10 d light drought after jointing, the effective rate of tiller of Ⅱ and more was decreased of severe drought. The number of spikes per unit area of Shannong 29 and Heng 0628 was decreased by 10.97%-11.52% and 15.00%-15.55%, respectively. The treatment of light drought at 0-5 d after jointing stage, the panicle characteristics, single stem grain yield and grain yield per unit area of the two cultivars were not significantly different from CK. The treatment of severe drought from 0 to 5 days after jointing, the fertile spikelets number and the grain number in the two cultivars were significantly reduced, and the main stem and later occurring tillers were not affected. The single grain weight of Shannong 29 was not affected, but the yield of single stem grain yield was significantly reduced, the single grain weight and single stem grain yield of Heng 0628 was significantly reduced, the grain yield per unit area of Shannong 29 and Heng 0628 decreased significantly, which was 5.14%-5.46% and 5.45%-6.24% lower than CK, respectively. For the light and severe drought from 0 to 10 days after jointing, the spikelets number, the fertile spikelets number, grain number, single grain weight, single stem grain yield and grain yield per unit area of the two cultivars were significantly reduced; The median tillers had substantial reduction; The number of grains per ear and the yield of single-stem grain and the grain yield per unit area of severely drought-treated stems were significantly lower than those of light drought treatment. Under the T3 condition, the grain yield per unit area of Shannong 29 and Heng 0628 was decreased by 12.87%-13.30% and15.52%-16.59%, respectively. Under the T4 condition, the grain yield per unit area of Shannong 29 and Heng 0628 decreased by 23.18%-25.92% and 26.05%-31.22%, respectively.【Conclusion】The above results indicated that short-term light drought after jointing (0-5 days after jointing, maintaining soil relative water content of 65%-70% and soil effective water content of 33.4-37.8 mm) had no significant effects on wheat earing and fruiting; The percent of tillers to panicle, the spikelets number, the fertile spikelets number, grain number, single grain weight and single stem grain yield of the low tillers (Ⅲ, Ⅰp) would be greatly reduced, and the grain yield per unit area was significantly reduced increased drought stress and time. However, the effective spike rate of high tillers (Ⅱp and Ⅰ1) in two degrees of 0-5 d after jointing and light drought training of 0-10 d after jointing was increased to some extent, which could make up for the loss caused by drought, which might be related to the decrease of the effective spike rate of low tillers, the decrease of population size, and the increase of nutrition and living space for high tillers. Shannong 29 had the stronger potential to adapt drought stress at jointing stage than Heng 0628.

The Regulation and Evaluation Indexes Screening of Chemical Topping on Cotton’s Plant Architecture

ZHU LingXiao,LIU LianTao,ZHANG YongJiang,SUN HongChun,ZHANG Ke,BAI ZhiYing,DONG HeZhong,LI CunDong

Scientia Agricultura Sinica. 2020, 53(20): 4152-4163. doi:10.3864/j.issn.0578-1752.2020.20.005

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【Objective】Plant architecture is of major agronomic importance because it strongly influences the suit-ability of a plant for cultivation, its overall yield and its economic coefficient. This study was aimed to explore the effects of chemical topping on the traits of cotton’s plant architecture and to make a comprehensive analysis, thus providing a theoretical basis for the application and popularization of chemical topping. 【Method】Three topping treatments, including manual topping, chemical topping and non-decapitation treatment, were established by using Jimian863 and Nongda601, which were widely grown in Yellow River Valley, in Hebei Agriculture University experimental base during 2015-2016, yield components, plant height, stem diameter, number of fruit branches and other plant architecture related indicators were measured, carry out research on the effect of chemical topping on plant architecture. 【Result】There was no significant difference in seed cotton yield between manual topping treatment and chemical topping treatment, and both treatments were significantly higher than that of non-decapitation treatment. Compared with non-decapitation treatment, chemical topping increased the seed cotton yield of Jiman863 and Nongda601 by 7.19% and 6.78%, respectively. Compared with non-decapitation treatment, chemical topping significantly decreased the cotton plant, internode number, fruiting branches number and fruiting node number, and significantly increased the diameter ratio of near and far branches. Chemical topping significantly decreased the internode length of stem and upper fruiting branches length. The effect of chemical topping on cotton’s plant architecture could be evaluated by the plant height and the diameter ratio of near and far branches according to Pearson correlation analysis, principal component analysis and grey correlation analysis. 【Conclusion】Chemical topping treatment had the same purpose on the regulation of cotton’s vegetative and regenerative growth as manual topping treatment, and had no significant effect on seed cotton yield. The plant height and the diameter ratio of near and far branches were taken as the main evaluation indexes for the regulation of chemical topping on cotton’s plant architecture.

Analysis on the Adaptability of Northward Planting of Brassica napus

CAO XiaoDong,LIU ZiGang,MI WenBo,XU ChunMei,ZOU Ya,XU MingXia,ZHENG GuoQiang,FANG XinLing,CUI XiaoRu,DONG XiaoYun,MI Chao,CHEN QiXian

Scientia Agricultura Sinica. 2020, 53(20): 4164-4176. doi:10.3864/j.issn.0578-1752.2020.20.006

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【Objective】To explore the ecological adaptability of breeding new varieties/lines with strong cold resistance and the feasibility of northward migration of Brassica napus (Brassica napus L.) at high altitude and high latitude, so as to realize the replacement of inferior and inefficient winter rape varieties, farmland ecological improvement and industrial promotion.【Method】Ten cold-resistant winter rape lines of Brassica napus were used as materials, and the original planting area and typical northward migration ecological region of winter rape of Brassica napus were selected as experiments to study the changes of growth period, yield, quality, plant type and other characters of cold-resistant winter rape varieties of Brassica napus in cold and cool areas at high latitude and high altitude.【Result】The results showed that Brassica napus-type winter rape could survive the winter safely in the original planting area (Tianshui), and the over-wintering rate of Brassica napus decreased obviously after moving northward. The control varieties（Ganza 1）could not survive the winter normally in the northward migration area (Baiyin and Dingxi); The 10 strong cold-resistant varieties could survive the winter safely in the Baiyin pilot, and the over-wintering rate was between 81% and 94%; The Dingxi pilot, the over-wintering rate was between 51% and 88%, of which 6 strong cold-resistant varieties could survive the winter safely in Dingxi (≥75%). The growth period of Brassica napus was prolonged by 52 days and 70 days after moving northward; The yield of cold-resistant lines move northward to Baiyin varied from-11.05% to 44.67%, and the highest yield was 5 170.80 kg·hm^-2 .The yield of all the tested lines in Baiyin decreased significantly, and the highest yield was 3 392.85 kg·hm^-2 .After planting northward, the plant height, branching position decreased, the secondary branch increased, the plant type became worse, the number of pods per plant decreased, the number of pods and 1000-grain weight increased, and the characters of pods became better. After moving northward, the contents of oil and glucosinolates in Brassica napus decreased, while the contents of erucic acid increased. One of the tested lines/species was a double-low, high-quality and strong cold-resistant strain (16NS20H1), and the other was a cold-resistant variety with low erucic acid (Ganza 1). Correlation analysis showed that overwintering rate, yield, plant height, branch position, main inflorescence length, oil content and glucosinolate content were significantly negatively correlated with altitude and latitude. It was positively correlated with annual average temperature, frost-free days and coldest monthly temperature, while the growth period, secondary branches changed to the contrary. In the northward moving area, the lodging resistance of Brassica napus type was significantly better than that of Brassica campestris L.. 【Conclusion】The cold resistance of the new cold-resistant winter rape strain of Brassica napus was obviously stronger than that of the control (Ganza 1), and it could survive the winter safely in the area of 36°12'N latitude and 2 248 m above sea level, and the winter rape lines with high yield, high quality and strong cold resistance could be selected and improve the yield level, quality and nutritional value of winter rape in the strong winter region of North China.

Effect of Panicle Neck Blast on Grain Yield and Stem Node Metabolites at the Rice Filling Stage

PAN ZhengYan,LIU Bo,JIANG HongBo,YAO JiPan,BAI YuanJun,XU ZhengJin

Scientia Agricultura Sinica. 2020, 53(20): 4177-4188. doi:10.3864/j.issn.0578-1752.2020.20.007

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【Objective】The objective of this study is to detect the stem node metabolite of susceptible cultivars which suffered the rice blast under the natural condition, analyze the reason for rice cultivars susceptibility and yield reduction, and to provide a theoretical basis for the susceptible mechanism study of japonica rice and ecological control.【Method】With susceptible cultivar which suffered the rice blast Liaoxing1 and japonica rice restorer C418 as the materials, the yield and quality character including seed setting rate, 1000-grain weight, length, brown rice were measured at the maturation stage. The incidence of panicle neck blast was investigated at the filling stage, and GC-MS was carried out to analyze the metabolites of diseased stem nodes and normal stem nodes, the metabolite composition between diseased stem nodes and normal stem nodes was analyzed by principal components analyses (PCA), the variable weights (VIP>1.0) of partial least-squares discriminant analysis (PLS-DA) were used to screen the species of differential metabolites, the difference of carbohydrate and amino acid metabolites was compared, and the reasons for the decrease of rice yield were analyzed from the metabolites.【Result】The incidence of panicle neck blast of Liaoxing1 and C418 was 57.23% and 82.94%, respectively. The panicle neck blast significantly reduced the head rice rate of the primary branch, the seed set rate, 1000-grain weight, length-width ratio and head rice rate of the secondary branch, there was no significant difference in other characters. 358 metabolites were identified in the two cultivars, the metabolites of diseased stem node increased by 73.46% and 67.88%, respectively. The PCA and PLS-DA analysis could clearly identify the metabolites of diseased stem node and normal stem node. The metabolite phenotypes of Liaoxing 1 and C418 were obviously different between the diseased stem node and normal stem node. Compared with normal stem node, the relative content of 61 metabolites induced by Liaoxing 1 diseased stem nodes changed significantly, and the relative content of 47 metabolites induced by C418 diseased stem nodes changed significantly, there were 30 metabolites in same of the diseased stem nodes of these two cultivars. The relative content of secondary metabolites of vanillic acid, alpha tocopherol, dehydroascorbic acid, triacontanol, daidzein and N-acetyl-d-mannosamine significantly decreased by 87.03%, 96.29%, 77.95%, 74.56%, 86.48% and 82.68% in Liaoxing1, respectively. In C418, it decreased by 99.92%, 99.68%, 97.26%, 86.67%, 94.96% and 76.74%, respectively. The relative content of the above six types of secondary metabolites in normal stem node of Liaoxing1 was 2.64, 2.39, 2.39, 2.27, 3.09 and 2.56 times of that in C418, respectively. The panicle neck blast increased 63.33% of carbohydrate metabolites and 68.42% of amino acid metabolites in stem node, the carbohydrate and amino acid metabolites increased by 1.02-17.92 and 1.05-13.12 times, respectively. Among them, the enhanced amplitude of carbohydrate metabolites in C418 was more significant.【Conclusion】The occurrence of panicle neck blast significantly changed the metabolite composition of susceptible cultivars Liaoxing 1 and C418 stem node, the susceptible cultivar may adjust its defensive reaction to the rice blast following the relative amount change of vanillic acid, alpha tocopherol, dehydroascorbic acid, triacontanol, daidzein and N-acetyl-d-mannosamine. By accumulating a large amount of the carbohydrate and amino acid metabolites, the diseased stem nodes reduce their transport to grains and restrain the formation of yield.

Identification of Rboh Family and the Response to Hormone and Citrus Bacterial Canker in Citrus

QIN XiuJuan,QI JingJing,DOU WanFu,CHEN ShanChun,HE YongRui,LI Qiang

Scientia Agricultura Sinica. 2020, 53(20): 4189-4203. doi:10.3864/j.issn.0578-1752.2020.20.008

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【Objective】Rboh is the main producer of reactive oxygen species (ROS), which is the key molecule involved in plant disease resistance signaling. To clarify the relationships between CsRboh family and citrus bacterial canker (CBC), the Rboh family of Citrus sinensis was identified and the bioinformatics characteristics as well as the expression patterns induced by biotic stress-related plant hormones and Xanthomonas citri subsp. citri (Xcc) were analyzed. It is also aimed to lay the foundation for further research of citrus Rboh genes in molecular breeding.【Method】Firstly, the RedOxiBase database and the citrus (sweet orange) CAP database were used to identify the citrus Rboh family sequences from the whole genome of C. sinensis. Then, series of bioinformatics tools were used to perform analysis of physical and chemical properties, phylogenetic relationships, chromosomal localizations, gene structures, protein functional domains, the conserved motifs and promoter elements systematically. Finally, qRT-PCR was used to analyze the expression patterns of CsRbohs induced by biotic stress-related plant hormones (salicylic acid, SA; jasmonic acid, JA; abscisic acid, ABA) and Xcc.【Result】A total of 7 members of the Rboh family in C. sinensis genome were identified (CsRboh01-CsRboh07). These genes encoded proteins contain 784-946 amino acid (AA) residues with isoelectric points (PI) ranging from 8.67 to 9.40. CsRbohs mainly distribute on the cell membrane system and cytoplasmic. The CsRbohs family can be divided into I to IV sub-groups based on the phylogenetic tree. These 7 genes are unevenly distributed on 5 sweet orange chromosomes. Each of the CsRbohs contains 4 typical functional domains, namely the respiratory burst NADPH oxidase domain (NADPH_Ox), the ferric reductase like transmembrane component (Ferric_reduct), the FAD binding domain (FAD_binding) and the ferric reductase NAD binding domain (NAD_binding). The promoter regions of CsRboh family genes contain different hormone response elements with different numbers and types. qRT-PCR showed that the expression of CsRboh gene family members in susceptible variety Wanjincheng and resistant variety Calamondin had some differences induced by different hormones and Xcc. According to the findings from phylogeny, homologous analysis and cis-acting elements analysis, CsRboh02, CsRboh04 and CsRboh06 had different expression patterns and levels induced by Xcc in resistant variety Calamondin and susceptible variety Wanjincheng.【Conclusion】CsRboh02, CsRboh04 and CsRboh06 may be closely related to the resistance and sensibility of citrus varieties, and they are three potential candidate genes for molecular breeding of resistance to canker disease, and the CsRboh family genes are preliminarily determined to play a key role in the process of citrus canker response.

Effects of Secondary Metabolites in Wheat Kernels on Activities of Three Detoxifying Enzymes and Related Gene Expression in Sitodiplosis mosellena

CHEN Rui,GAO He,ZHANG GuoJun,ZHU KeYan,CHENG WeiNing

Scientia Agricultura Sinica. 2020, 53(20): 4204-4214. doi:10.3864/j.issn.0578-1752.2020.20.009

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【Objective】The objective of this study is to clarify the relationship between secondary metabolite contents at grain filling stage of wheat and detoxification enzyme activities of glutathione S-transferase (GST), carboxylesterase (CarE) and cytochrome P450 (CYP450) and related gene expression in Sitodiplosis mosellena, and to explore the role of secondary metabolite in wheat resistance to S. mosellena and the metabolic mechanism.【Method】The numbers of S. mosellana larvae in wheat kernels of four resistant and four susceptible wheat varieties (lines) planted in experimental fields in Zhouzhi County, Shaanxi Province were investigated by dissecting wheat ears in May 2016. Wheat kernels at filling stage from eight varieties (lines) and S. mosellena larvae feeding wheat kernels of eight varieties (lines) were simultaneously collected. The contents of ferulic acid, tannin, total phenol and total flavonoid were determined by colorimetry, vanillin assay, Folin-Ciocalteu assay and sodium nitrite-aluminium nitrite assay, respectively. The activities of GST, CarE and CYP450 and expression levels of GST1, CarE2 and CYP6A1 in collected S. mosellena larvae were determined using enzyme linked immunosorbent assay (ELISA) and qPCR methods, respectively. And then, the relationship between secondary metabolite contents in various wheat varieties (lines) and resistance indictors of wheat to S. mosellena, as well as the interaction of secondary metabolites with detoxification enzyme activity and gene expression in S. mosellena larvae were studied by correlation analysis.【Result】The contents of ferulic acid, tannin, total phenol and total flavonoid of wheat kernel at filling stage of eight wheat varieties (lines) were significantly different. The highest contents were in the resistant variety Kenong 1006 for ferulic acid and tannin, in the resistant variety Jinmai 47 for total phenol, and in the susceptible variety Xinong 88 for total flavonoid. The activities of GST, CarE and CYP450 and expression levels of GST1, CarE2 and CYP6A1 in S. mosellena larvae from eight wheat varieties (lines) were also significantly different. The highest enzyme activities were in larvae from resistant varieties Kenong 1006 and Shaanmai 139 for GST and CarE, respectively, and from the susceptible variety Xiaoyan 6 for CYP450. The expression levels of GST1 and CarE2 were higher in larvae feeding four resistant varieties than those feeding four susceptible varieties, but CYP6A1 expression level had not obvious regularity between larvae feeding resistant and susceptible varieties. Correlation analysis results showed that the ferulic acid content of wheat kernel had significant negative correlation (P<0.05) with the four resistance indicators of wheat to S. mosellena including percentage of infested ears, percentage of infested grains, insect number per ear and estimated loss rate. There was no significant correlation between the contents of tannin, total phenol and total flavonoid and the four resistance indicators. The GST and CarE activities of S. mosellena feeding wheat kernels were significantly positively correlated to the ferulid acid content of wheat kernels, and the CYP450 activity of S. mosellena was significantly negatively correlated to the tannin content (P<0.05). No significant correlation was observed between the total phenol and total flavonoid contents of wheat kernels and the activity of any of the three detoxification enzymes. The expression levels of S. mosellena GST1 and CarE2 were significantly positively correlated with the activities of GST and CarE (P<0.05), respectively, while S. mosellena CYP6A1 expression level was not significantly related with the activity of CYP450.【Conclusion】Ferulic acid is the main secondary metabolite of wheat anti-S. mosellena, and can induce GST and CarE activities of S. mosellena larvae and related gene expression. GST and CarE jointly participate in the detoxification metabolism and adaptation of S. mosellena to wheat secondary metabolites.

Effects of Lime and Straw Addition on SOC Sequestration in Tier Soil

CAO BinBin,ZHU YiHui,JIANG YuHan,SHI JiangLan,TIAN XiaoHong

Scientia Agricultura Sinica. 2020, 53(20): 4215-4225. doi:10.3864/j.issn.0578-1752.2020.20.010

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【Objective】Soil organic carbon (SOC) sequestration is crucial for improving soil fertility and agricultural production sustainability. The soil inorganic carbon (SIC) is closely related to SOC with regarding with inter-transformation, which has also great effect on carbon sequestration. Crop straw return has been recognized as one of the most important organic amendment improving soil organic carbon sequestration in farmland. Meanwhile, the addition of lime also contributes greatly to increasing SIC, thereby affecting the SOC sequestration. However, the mechanism of simultaneous incorporation of crop straw and lime affecting on the CO₂ emission, SOC and SIC dynamics are not well understood, and how the SOC sequestration responds to the initial level of SOC is not clear, particularly after straw return. 【Method】 The incubation experiment and stable isotope technique (¹³C) were used in the study. The two tested soils were collected from a field with continuously cropping of winter wheat for 16 years, which was subjected to differential crop residue and nitrogen managements over long-term, including (1) S₀N₀ soil (no straw return+ nitrogen fertilizer application: 0); (2) S₁N₁ soil (high amount of straw return+ nitrogen fertilizer application: 240 kg·hm^-2). And then the two soils were both incubated with or without addition of straw and lime for 120 days under 25℃. 【Result】 The study showed that the soil cumulative CO₂ emission was observed 42.9% higher in S₁N₁ soils than that in S₀N₀ soils, when without straw and lime addition. In both soils, the straw addition alone increased the soil cumulative CO₂ emission by averages of 81.6% and 70.4%, respectively, compared with straw absence. Meanwhile, the increase of the cumulative CO₂ emission in S₀N₀ soils was higher than in S₁N₁ soils. This showed that straw addition had a greater impact on the native OC mineralization in soil with low initial SOC. Lime addition decreased soil cumulative CO₂ emission in both soils whether straw addition or not. Straw addition increased the SOC by 2.95 g·kg^-1 and 3.19 g·kg^-1 in S₀N₀ soils and S₁N₁ soils, respectively, while reduced the SOC by 1.36 g·kg^-1 in S₁N₁ soils and did not affect the SOC in S₀N₀ soils when combining with lime addition. Using¹³C stable isotope technology, the newly formed organic carbon (OC) in the two soils both significantly increased after straw addition, therein which increased 25.8% in S₀N₀ soils when compared with S₁N₁ soils. However, the conjoint addition of lime and straw did not modify the newly formed SOC when compared with the addition of straw alone, which showed that the lime addition had no effect on the decomposition process of straw in the soil. Over all, straw addition alone increased the SOC net sequestration by 3 066.3 mg?kg^-1and 2 480.53 mg?kg^-1 in S₀N₀ soils and S₁N₁ soils, respectively. The conjoint addition of lime and straw had no significant effect on the SOC net sequestration in S₀N₀ soils, but there was a decreasing trend on the SOC net sequestration in S₁N₁ soils. Lime addition reduced the cumulative CO₂ emission by 469 mg?kg^-1 and 529 mg?kg^-1 in S₀N₀ soils and S₁N₁ soils, respectively, which approximately equaled to the increases in SIC (by 443 mg?kg^-1 and 566 mg?kg^-1, respectively).【Conclusion】 In conclusion, the soil with low initial SOC content had higher potential in SOC sequestration. Lime addition might be an effective method to affect soil carbon sequestration and reduce soil CO₂ emission through chemical reactions.

Soil Ecoenzymatic Stoichiometry and Relationship with Microbial Biomass in Fluvo-Aquic Soils with Various Fertilities

ZHANG Lu,ZHANG ShuiQing,REN KeYu,LI JunJie,DUAN YingHua,XU MingGang

Scientia Agricultura Sinica. 2020, 53(20): 4226-4236. doi:10.3864/j.issn.0578-1752.2020.20.011

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【Objective】 Microorganisms and soil enzymes are important drivers for nutrient cycles in farmland soil. To improve the soil fertility and apply fertilizer reasonably, it’s essential to understand the ecoenzymatic stoichiometry of carbon : nitrogen : phosphorus (C : N : P) and quantify its relationship with microbial biomass in soils with various soil fertility levels.【Method】Soil samples were collected from five farmer fields, including high, medium and low fertility levels. Soil type was fluvo-aquic soil. The contents of microbial biomass C and N (SMBC and SMBN), activities of five enzymes and integrated enzyme index (IEI) were analyzed. The enzymes included C-related: β-1, 4-glucosidase (BG) and Cellobiohydrolase (CBH); N-related: β-1, 4-N-acetylglucosaminidase (NAG) and leucine aminopeptidase (LAP); P-related: alkaline phosphatase (AP). The stoichiometry of the enzymes and its relationship with corresponding microbial biomass were studied. 【Result】 The SMBC of high fertility soil was 2.6 and 5.8 times of that in medium and low fertility soil, respectively, and SMBN in high fertility soil was about 3.1 and 5.5 times as much as that in medium and low fertility soils, respectively. There was no significant difference for SMBC/SMBN among the soils. All the five enzyme activities and IEIfollowed the same trend among soils: high fertility > medium fertility > low fertility, and there was a positive linear correlation between C-related/N-related enzymes with SMBC/SMBN. The slope of the relationship indicated that the activity of (BG + CBH) and (NAG + LAP) were increased by 0.134 and 10.53 nmol·g^-1·h^-1 when SMBC and SMBN were increased by 1 mg·kg^-1, respectively. The ratio were lower than 1 both for ln (BG+CBH) : ln (NAG+LAP) and ln (BG+CBH) : ln AP, indicating that the primary limiting factor for nutrient transformation was carbon resource in fluvo-aquic soil. The C : N : P stoichiometry of enzymes in high fertility soil was closer to optimum value than that in low fertility soil.【Conclusion】Application of organic materials, such as manure and straw, would be an efficiency management strategy to promote nutrient cycling by supplement C resources in fluvo-aquic soil. Appropriate ecoenzymatic C : N : P stoichiometry might contribute the high yield and nutrient use efficiency in high fertility soil.

Effect of Maize Straw Return Aftereffect on Nitrogen Use Efficiency of Maize

ZOU WenXiu,HAN XiaoZeng,LU XinChun,CHEN Xu,HAO XiangXiang,YAN Jun

Scientia Agricultura Sinica. 2020, 53(20): 4237-4247. doi:10.3864/j.issn.0578-1752.2020.20.012

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【Objective】Maize straw return has been one of agronomic practices for improving soil fertility. The effect of straw return aftereffect on Nitrogen use efficiency (NUE) was considered, with the objective of suppling the theoretical guidance for proposing the straw return patterns being favorable to enhance the NUE.【Method】Based on the black soil with heavy clay content in the Hailun agro-ecosystem experimental station of Chinese Academy of Sciences, the micro-plot experiment was carried out using ¹⁵N isotope techniques in 2016 under the treatment of the straw return (10 000 kg·hm^-2) only once established in 2011. Seven treatments were set up, including control without maize straw return (CK), maize straw cover (D₀), maize straw incorporated within 0-20 cm soil depth (D_0-20), 0-35 cm soil depth (D_0-35), 20-35 cm soil depth (D_20-35), respectively, as well as maize straw placed on the 35 cm depth (D₃₅) and 50 cm depth (D₅₀).【Result】The aftereffect of maize straw return increased NUE by enhancing the cumulation of dry biomass and nitrogen in maize organs. The maize dry biomass shown in the decreasing trend of D_0-35>D_20-35>D_0-20>CK≥D₀>D₃₅>D₅₀, D_0-35 and D_20-35 treatments (Maize straw incorporated within deep soil) significantly increased by 7.1%-47.7% and 2.0%-39.1% (with exception of leaf), respectively (P<0.05), compared with other treatments. The different treatments didn’t impact the nitrogen (N) contents of maize organs, but D_0-35, D_20-35 and D_0-20 treatments significantly increased the N cumulation in maize organs (P<0.05) with the increase of 15.8%-20.2%, 8.5%-18.2% and 27.9%-39.5%, respectively (P<0.05), compared with CK, D₀, D₃₅, and D₅₀. The cumulation of ¹⁵N under D_0-35and D_20-35treatments significantly increased by 5.1%-38.4% and 9.3%-31.8%, respectively, compared with other treatments. ¹⁵N of more than 74.1% were accumulated in the grain, different treatments didn’t impact the ¹⁵N distribution in maize organs, indicating that NUE was increased by improving the N uptake of whole maize. The NUE and N retention rate reached the largest value under D_0-35 with the increase of 1.9-12.7 percentage and 6.9-21.2 percentage, respectively, However, the N loss under D_0-35 reached the lowest value with the decrease of 8.8-31.3 percentage. D₀, D₃₅ and D₅₀ treatments (straw return in some soil layer) didn’t significantly increased the NUE compared with CK treatment, meanwhile the N loss was higher 3.6 and 4.4 percentages than that in CK treatment. The result indicated that straw return in some soil layer had potential risk in the increase of N loss, constructing fertile cultivated soil layer by straw incorporation within deep soil depth was effective agronomic practice with high NUE. The correlation analysis demonstrated that root biomass, light fraction organic carbon and soil physical properties contributed the increase of NUE in study site. N contribution rate was significantly higher 3.74, 4.26, 3.79 and 4.51percentages in D_20-35, D₃₅ and D₅₀ treatments than that in CK treatment, no significantly difference was observed among treatments with straw return. 【Conclusion】NUE could be increased by enhancing soil fertility and improving soil structure under maize straw incorporated within deep soil, and constructing fertile soil layer in black soil with heavy clay content.

Effects of End of Day Far-Red Light on Growth, Histiocyte Morphology and Phytohormones Content of Pumpkin Seedlings

LIU Qi,MEI YanHao,LI Qi,MA HongXiu,WU YongJun,YANG ZhenChao

Scientia Agricultura Sinica. 2020, 53(20): 4248-4258. doi:10.3864/j.issn.0578-1752.2020.20.013

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【Objective】This study was aimed to understand the effects of growth, histiocyte morphologic and phytohormone levels of pumpkin seedlings under different doses of End-Of-Day Far-Red light (EOD-FR) treatment, so as to provide a theoretical basis for the application of far-red in agricultural industry. 【Method】 Taken the pumpkin (cv. Japanese cedar) as experimental material, the growth state of hypocotyl’s histiocyte morphology and the contents of auxin (IAA), zeatin (ZT), gibberellin (GA₃) and brassinolide (BR) were measured under 0 (CK), 2 (T1) , 4 (T2) , 6 (T3) , 8 (T4) , 10 (T5) and 12 (T6) mmol·m^-2·d^-1dose far-red light treatments, respectively. 【Result】 The hypocotyl length and plant height of pumpkin seedlings were significantly increased by EOD-FR treatment, while there was no significant effect on the diameter, dry and fresh weight of pumpkin stem. Compared with the control group, the lengths of hypocotyl’s parenchyma cell of each treatment were significantly increased by 34.6%, 20.7%, 31.3%, 25.6%, 32.8% and 20.9%, respectively; the collenchyma's thickness were significant increased by 19.6%, 22.4%, 21.2%, 23.9%, 19.6% and 28%, respectively. After EOD-FR treatment, the contents of IAA in roots of pumpkin seedlings significantly increased, and the contents of IAA, GA₃ and ZT in hypocotyls, IAA, GA₃ and BR in cotyledons, IAA and BR in euphylla were also increased. 【Conclusion】Through increasing the hormone levels under EOD-FR treatment, the histiocyte morphology could be changed, and the hypocotyl elongation could be increased.

Whole-Genome Identification and Expression Analysis of SPL Gene Family in Dimocarpus Longan

LU BaoShun,ZHU YongJing,ZHANG ShuTing,LÜ YuMeng,LI XiaoFei,SONG YuYang,LAI ZhongXiong,LIN YuLing

Scientia Agricultura Sinica. 2020, 53(20): 4259-4270. doi:10.3864/j.issn.0578-1752.2020.20.014

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【Objective】To provide a reference for the study of the function of SPL in Dimocarpus longan growth and development, all members of the longan SPL(DlSPL) gene family were identified and their expression patterns were analyzed in this study.【Method】The DlSPL family members, with their basic physicochemical properties, gene structures, phylogenetic relationships, and cis-acting promoter elements, were identified by bioinformatics analysis, and their expression patterns in non-embryonic callus (NEC), embryogenic culture and embryonic callus (EC) under different hormone treatments were analyzed by FPKM and qRT-PCR technology. 【Result】The results showed that there were 14 DlSPL gene family members, and their gene and protein structures were all highly conserved and contained only one SBP domain. The promoters of DlSPL genes had a large number of light response elements, stress response elements, hormone response elements, tissue specific regulatory elements and cis-regulatory elements related to plant growth and development, indicating that DlSPL genes might be regulated by light, hormone, stress and other factors. RNA-seq expression analysis showed that 11 of 14 DlSPL members were detected in EC under the different light qualities and different hormones treatments, and only DlSPL8 showed a down-regulated expression trend under blue and white light treatments, and the expression levels of DlSPL3 and DlSPL13 members were higher in EC under 2, 4-D+KT treatment than that of under 2, 4-D and KT treatment, respectively; 13 DlSPLs were detected in non-embryogenic and embryogenic cultures, seven members of them had the highest expression levels in the NEC stage. QRT-PCR results showed that DlSPL1 and DlSPL14 had the highest expression levels in the EC stage, DlSPL5, DlSPL7 and DlSPL13 had the highest expression levels in the incomplete embryonic compact structure (ICpEC) stage; DlSPL1, DlSPL5, DlSPL7, and DlSPL13 were down-regulated under abscisic acid treatments. DlSPL5 was up-regulated under methyl jasmonate acid treatments, while the other three members were down-regulated. 【Conclusion】 A total of 14 longan SPL gene family members contained a highly conserved SBP domain were identified, and the DlSPL gene family might play an important role in longan somatic embryos, and respond to abscisic acid and methyl jasmonate element treatments.

Effects of Potassium Application Levels on Nutrient, Yield and Quality of Newhall Navel Orange

ZHANG Ji,LI JunJie,WAN LianJie,YANG JiangBo,ZHENG YongQiang,LÜ Qiang,XIE RangJin,MA YanYan,DENG Lie,YI ShiLai

Scientia Agricultura Sinica. 2020, 53(20): 4271-4286. doi:10.3864/j.issn.0578-1752.2020.20.015

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【Objective】This study investigated the effects of different potassium application levels on nutrient absorption, fruit yield and quality and determined the appropriate amount of potassium application, which provided a theoretical basis for the scientific application of potassium fertilizer for Newhall navel orange.【Method】Six different potassium application levels, including K0 (0 K₂O), K1 (0.38 kg/plant), K2 (0.64 kg/plant), K3 (0.89 kg/plant), K4 (1.28 kg/plant) and K5 (1.40 kg/plant), were performed to study its effects on nutrient absorption of branches, leaves and fruit, fruit quality and yield by using 7-year-old Newhall navel orange grafted on Poncirus trifoliata (L.) .【Result】The dry matter accumulation of leaves and branches of Newhall navel orange in different periods were similar among different groups, while spring shoots were higher than autumn shoots as well as leaves higher than branches. Dry matter quality of leaves and branches were increased first and then decreased with the increased potassium level. The nitrogen fertilizer enhanced the nutrient absorption of nitrogen, phosphorus and potassium in spring shoots with a peak level under K2 treatment, and it also similarly increased the absorption of nitrogen, phosphorus and potassium in autumn shoot with lower level in K5 group than that in K0 group. Potassium had no significant effect on nitrogen, phosphorus and potassium content of fruit. Maximum nitrogen removal was found under K3 treatment, and then phosphorus and potassium were found under K2 treatment. The amount of fruit nutrient removal was nitrogen ≈ potassium > phosphorus. Potassium application first enhanced the yield and single fruit weight, and then decreased, and the highest level of yield under K2 treatment was higher than that under K0 treatment. Potassium application made the fruit redder with the best effect under K3 treatment. Potassium application first thinned the pericarp and then thickened with the thinnest level under K2 treatment. Potassium application first enhanced the vitamin C content, and then decreased with the highest level under K3 treatment. The maximum soluble sugar content was found under K3 treatment, and the minimum titratable acid content was found under K2 treatment. Potassium application first enhanced the soil organic matter and available N, and then decreased at the highest level under K1 and K3 treatment, respectively. Potassium application first decreased the soil available P, and then enhanced at the highest level under K4 treatment, and then decreased. Potassium application under K4 treatment could maximize the content of available potassium in the soil, and the most of the available potassium was distributed in the surface soil (0-20 cm) and tended to accumulate in the deep soil (60—80 cm). Correlation analysis showed that the potassium content in spring leaves was significantly positively corrected with the total nitrogen and potassium content of fruit and was significantly negatively correlated with fruit firmness. The potassium content in autumn shoots was significantly positively correlated with yield and TSS content, and the potassium content in autumn twigs was significantly negatively correlated with the thickness of peel. The soil available K content was significantly negatively correlated with the total nitrogen and potassium content of fruit, and was significantly negatively correlated with fruit hardness.【Conclusion】These results indicated that the pure potassium oxide application rate was 0.64 to 0.89 kg/plant to ensure high level of yield and fruit quality, and to facilitate nutrient absorption and utilization of Newhall navel orange tree while maintaining high soil fertility. At the same time, the risk of soil potassium accumulation and pollution was reduced.

Effects of PSMB5 on the Proliferation and Myogenic Differentiation of Skeletal Muscle Satellite Cells

LAI YuTing,ZHU FeiFei,WANG YiMin,GUO Hong,ZHANG LinLin,LI Xin,GUO YiWen,DING XiangBin

Scientia Agricultura Sinica. 2020, 53(20): 4287-4296. doi:10.3864/j.issn.0578-1752.2020.20.016

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【Objective】 The aim of this study was to investigate the effects of proteasome subunit beta type-5(PSMB5) on the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells. 【Method】 In this study, the in vitro induced myogenic differentiation model of bovine skeletal muscle satellite cells was used to simulate the growth and development of bovine skeletal muscle. Firstly, the expression of PSMB5 in bovine skeletal muscle satellite cells before and after the differentiation was detected. The mRNA expression level of PSMB5 was detected by qRT-PCR, and the protein expression level of PSMB5 was detected by western blotting. Then, the small interfering RNA si-RNA-PSMB5 (si-PSMB5) was designed and synthesized, and PSMB5 overexpression plasmid pcDNA3.1-PSMB5 (pcDNA-PSMB5) was constructed, which were transfected into bovine skeletal muscle satellite cells by lipofectamine 3000, and the transfection effect was detected by qRT-PCR and Western blotting. Finally, EdU staining was used to detect the effects of PSMB5 interference and overexpression on the proliferation of bovine skeletal muscle satellite cells. Further, in vitro myogenic differentiation of bovine skeletal muscle satellite cells was induced, and the myotube formation of bovine skeletal muscle satellite cells was observed under light microscope. The expression of myosin heavy chain (MyHC) protein, a marker of differentiation, was detected by western blotting. The effects of PSMB5 on proliferation and differentiation of bovine skeletal muscle satellite cells were analyzed. 【Result】There was a significant difference in the expression level of PSMB5 before and after the differentiation of bovine skeletal muscle satellite cells. After the differentiation of bovine skeletal muscle satellite cells, the expression level of PSMB5 mRNA and protein was significantly higher than that in the proliferation period (P<0.05). When PSMB5 was interfered or overexpressed, there was no significant difference in EdU positive cell rate between the two groups (P>0.05). After interfering with the expression of PSMB5, the number of myotubes formed by cell differentiation was significantly less than that of the control group, and the protein expression level of MyHC was significantly lower than that of the control group (P<0.05); however, after overexpression of PSMB5, the number of myotubes formed by cell differentiation was significantly higher than that of the control group, and the protein expression level of MyHC was significantly higher than that of the control group (P<0.05). 【Conclusion】The results showed that the PSMB5 had no significant effect on the proliferation of bovine skeletal muscle satellite cells, but it had a significant regulatory effect on the myogenic differentiation process of bovine skeletal muscle satellite cells. Interference with PSMB5 expression could inhibit the myogenic differentiation process, while overexpression of PSMB5 could promote the myogenic differentiation process of bovine skeletal muscle satellite cells in vitro. In this study, the specific regulatory effect of PSMB5 on the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells was explored, which laid a foundation for further study on the regulatory mechanism of PSMB5 in bovine muscle myogenic differentiation.

Molecular Characterization of Tibetan Sheep BOLL and Its Expression Regulation and Functional Analysis in Testis

LI TaoTao,WANG Xia,MA YouJi,YIN DeEn,ZHANG Yong,ZHAO XingXu

Scientia Agricultura Sinica. 2020, 53(20): 4297-4312. doi:10.3864/j.issn.0578-1752.2020.20.017

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【Objective】 As a RNA-binding protein, BOLL exerts integral roles during spermatogenesis through interactions with other molecules. The present study was aimed to analyze the sequence characteristics of Tibetan sheep BOLL and its expression and distribution patterns in testis which, in turn, explored the regulation and potential biological function of BOLL expression, hoping to offer much needed perspective and molecular insight for deciphering the mechanism of BOLL during sheep spermatogenesis in the future. 【Method】Eight healthy male Tibetan sheep from each of three key developmental stages, including 3 months old (sexually immature), 1 year old (sexually mature) and 3 years old (adult), were selected. The full-length coding sequence (CDS) region of Tibetan sheep BOLL was cloned by real time PCR (RT-PCR) using total RNA from the right testis samples; the sequence and structural signatures of BOLL and its interacting proteins were analyzed via relevant bioinformatics software; the expression and immunolocalization characteristics of BOLL in testicular tissues at three developmental stages were assessed with quantitative RT-PCR (qRT-PCR), Western blot, and immunofluorescence staining; the competing endogenous RNA (ceRNA) regulatory network and GO functional annotation for ovine BOLL were investigated with the aid of related databases, based on previous data of an integrative analysis of transcriptional profiles from Tibetan sheep testicular tissues by our group, and their expression patterns and targeting relationships were verified by qRT-PCR and dual luciferase reporter assay, respectively. 【Result】 The full CDS region of Tibetan sheep BOLL was 888 bp in length, capable of encoding 295 amino acids which contained a RRM domain of 81 amino acids (near the N-terminal) and a DAZ repeat motif of 25 amino acids. Ovine BOLL exhibited high sequence homology and evolutionary conservation with other mammals, especially for goat, cattle, and yak. There was the potential interaction between sheep BOLL protein and 10 proteins associated with the development of male germ cells. With increasing age, mRNA expression of BOLL was consistently up-regulated in Tibetan sheep testes, while protein expression of which was up-regulated followed by down-regulation. BOLL protein predominantly existed in spermatids from post-pubertal (1 year old and 3 years old) testes, and small amounts were present in spermatocytes, as well as spermatogonia in testes throughout development stages. The qRT-PCR results showed that compared with 3 months old, the expression levels of microRNAs (miRNAs) oar-miR-127-5p、oar-miR-382-5p and oar-miR-760-3p exhibited an extremely significant reduction (P<0.01) , while the expression levels for long noncoding RNAs (lncRNAs) LOC105602204, LOC105603195 and LOC105616228, as well as for circular RNAs (circRNAs) circ-ECT2L and circ-SPHKAP exhibited an extremely significant increase in 1 year old and 3 years old (P<0.01). oar-miR-127-5p and oar-miR-760-3p significantly decreased the luciferase activity of BOLL 3′-UTR wild-type, and oar-miR-760-3p significantly decreased the luciferase activities of wild-type Circ-ECT2L and wild-type LOC105616228 reporter genes. 【Conclusion】 This study was the first to report the molecular characteristics and expression patterns of BOLL gene and regulation of its expression in Tibetan sheep testis. BOLL was predominantly expressed in the post-meiotic round and elongating spermatids of Tibetan sheep, and the expression of which was directly targeted and negatively regulated by oar-miR-127-5p and oar-miR-760-3p; and was positively regulated by oar-miR-760-3p -mediated circRNA Circ-ECT2L and lncRNA LOC105616228. In turn, which might may interact with downstream signaling molecules to participate in the differentiation of ovine spermatids into mature spermatozoa.

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