Scientia Agricultura Sinica

Research Progress of PPR Protein in Plant Abiotic Stress Response

LI Cheng, LU Kai, WANG CaiLin, ZHANG YaDong

Scientia Agricultura Sinica. 2023, 56(24): 4801-4813. doi:10.3864/j.issn.0578-1752.2023.24.001

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Abiotic stress is one of the main factors causing global grain yield reduction. It is of great significance to study the function and response mechanisms of plant stress-related proteins to improve crop stress resistance. Pentatricopeptide repeat (PPR) proteins, belong to the largest family of nuclear coding proteins in higher plants and are named because they contain highly specific PPR motifs. Depending on motif type and arrangement, PPR proteins can be classified as P and PLS, and PLS proteins can be further classified as PLS, E, E+, DYW, and other subclasses based on their carboxyl-terminal domains. PPR proteins are widely distributed in terrestrial plants, mainly in chloroplasts and mitochondria, and a few in the nucleus. As sequence-specific RNA binding proteins, PPR proteins are involved in multiple aspects of plant RNA processing, including RNA editing, splicing, stabilization, and translation. PPR protein plays a variety of important roles in the whole life process of plants, but the mechanism of its action in plant stress resistance is not well understood. Based on the localization and function of PPR proteins related to abiotic stress reported, the mechanism of PPR proteins involved in regulation of abiotic stress, including post-transcriptional regulation and retrograde signaling, was reviewed and discussed in this paper. Post-transcriptional regulation is related to the role of PPR proteins in the modification of RNA after transcription. It is generally believed that PPR affects stress resistance in plants by regulating the expression of stress-related genes via binding RNA and by regulating the metabolism of organelle RNA. In terms of retrograde signaling, damage to PPR proteins can lead to impaired mitochondrial or chloroplast function, and then produce various retrograde signals (such as ROS), thereby regulating the expression of related genes and resisting adversity. However, since plastid signaling is affected by many environmental factors, some of which are still unclear, the mechanism of the PPR protein in retrograde signaling remains to be clarified. In addition, PPR proteins are pleiotropic and some have important effects on plant growth and reproduction while acting on stress resistance. Finally, this paper further analyzed the current research status of PPR protein as an RNA editing tool, discussed the remaining problems and research prospects of PPR protein in the direction of abiotic stress, and pointed out the key points and difficulties that need to be paid attention to in future research, to provide references for further research on PPR protein and crop abiotic stress resistance breeding.

Identification and Genetic Analysis of QTL for Spike Length in Wheat

YAO QiFu, ZHOU JieGuang, WANG Jian, CHEN HuangXin, YANG YaoYao, LIU Qian, YAN Lei, WANG Ying, ZHOU JingZhong, CUI FengJuan, JIANG Yun, MA Jian

Scientia Agricultura Sinica. 2023, 56(24): 4814-4825. doi:10.3864/j.issn.0578-1752.2023.24.002

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【Objective】Spike length (SL) plays an important role in determining spike structure and yield potential of wheat. Quantitative trait loci (QTL) for spike length were excavated and their genetic effects were further analyzed to provide theoretical basis for molecular breeding. 【Method】This study consisted of a population of 198 F₆ recombinant inbred lines (RIL) derived from the cross between the natural mutant msf and the cultivar Chuannong 16 (MC population). The MC population and its parents were planted in five different environments including Wenjiang in 2021 and 2022 (2021WJ and 2022WJ); Chongzhou in 2021 and 2022 (2021CZ and 2022CZ); and Ya’an in 2021 (2021YA) for spike length measurement. The 16K SNP chip-based constructed high-quality and high-density genetic linkage maps were used to map QTL for spike length. Additionally, the genotype of the flanking markers for the major spike length QTL was used to analyze its genetic effect on yield-related traits and thus to evaluate its potentiality for yield improvement.【Result】A total of 14 QTL for spike length were identified and they were mainly distributed on chromosomes 1A (one), 1B (one), 2B (one), 3D (three), 4A (one), 4D (two), 5A (one), 5B (one), 7A (one), 7B (one), and 7D (one). Among them, QSl.sau.1A was detected in four environments and the best linear unbiased prediction (BLUP) value, explained 6.46% to 20.12% of the phenotypic variation, and thus was regarded as a major QTL. The positive allele at QSl.sau.1A came from the parental line msf. QTL analysis across multiple environments also detected QSl.sau.1A, indicating it exhibits minimal environmental influence and represents a major and stably expressed QTL. The effect of QSl.sau.1A was successfully verified in two populations with different genetic backgrounds. Genetic effects analysis showed that the positive allele of QSl.sau.1A showed a significant effect on improving grain number per spike (12.68%), grain weight per spike (14.99%), 1000-grain weight (5.79%), flag leaf width (2.94%), spikelet number (1.48%), and flowering date (0.61%), and a significant effect of reducing plant height (-6.47%) and effective tiller number (-36.11%).【Conclusion】A major and stably expressed spike length QTL, QSl.sau.1A, was detected on chromosome 1A. Its positive allele significantly increased grain number per spike, grain weight per spike, thousand grain weight, and spikelet number per spike, indicating its great breeding value.

Response and Drip Irrigation Re-Watering Compensation Effect of Spring Wheat Roots to Drought Stress with Different Drought Tolerance Varieties

WANG RongRong, CHEN TianPeng, YIN HaoJie, JIANG GuiYing

Scientia Agricultura Sinica. 2023, 56(24): 4826-4841. doi:10.3864/j.issn.0578-1752.2023.24.003

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【Objective】The objective of this study is to investigate the differences in morphological and physiological responses of root growth of drip irrigated spring wheat with different drought sensitivity to stage drought stress, to further elaborate the physiological mechanisms of drought resistance and water conservation in drip irrigated spring wheat in Xinjiang, and to provide a theoretical basis for further water-saving and high-yield in Xinjiang wheat region. 【Method】 From 2021-2022, the strong drought resistance variety Xinchun 6 and the weak drought resistance variety Xinchun 22 were used as the test materials by using the soil column cultivation method. Mild (T1 and T3, 60%-65% FC, FC is the field capacity) and moderate (T2 and T4, 45%-50% FC) drought stress treatments were conducted during the tillering and jointing stages, with conventional irrigation as the control (CK, 75%-80% FC), the effects of pre-reproductive drought stress on root morphological characteristics (root length density (RLD), root volume density (RVD)), antioxidant system (malondialdehyde (MDA), superoxide dismutase (SOD), peroxidase (POD)), osmoregulation substance (proline (Pro), soluble sugars (SS)) and spatial and temporal characteristics of root activity were studied. The compensatory effect of drip irrigated spring wheat root growth on drought-rehydration was analyzed.【Result】RLD and RVD showed a trend of increasing and then decreasing with increasing drought stress, and under mild drought (T1 and T3) conditions, RLD and RVD in the 20-60 cm soil layer were significantly increased. The MDA content in the root of each soil layer showed an upward trend with the intensification of stress, while SOD, POD, Pro, and SS all increased first and then decreased with the intensification of drought, and gradually increased with the deepening of the soil layer. After rehydration of T1 treatment, root morphological characteristics, antioxidant enzyme activity, osmotic substances and root activity all reached the maximum value, which in turn increased the yield by 2.77% to 19.58% compared to the rest of the treatments. Stepwise regression analysis showed that RVD and SS were important determinants of yield, RLD, SOD and SS were the most significant indicators of Xinchun 6 drought resistance; RLD, MDA and POD were the most significant indicators of Xinchun 22 drought resistance.【Conclusion】Spring wheat maintains 60%-65% FC during the tillering and jointing stages, after drip irrigation and rehydration, it increases the distribution proportion of roots in the 20-60 cm soil layer, enhances the root system’s ability to remove reactive oxygen species and osmotic adjustment, delays root senescence and improves root physiological characteristics, thus increasing yield.

Comparative Analysis of the Effects of Different Types of Plastic Film on Peanut Growth and Rhizobacterial Community

YU TianYi, YANG JiShun, WU ZhengFeng, ZHANG ZhiMeng, SHEN Pu, ZHENG YongMei, LI ShangXia, WU JuXiang, SUN QiQi, WU Yue

Scientia Agricultura Sinica. 2023, 56(24): 4842-4853. doi:10.3864/j.issn.0578-1752.2023.24.004

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【Objective】This study was carried out to investigate the effects of different types of plastic film on peanut rhizobacterial community and peanut growth/yield, explore the relationships between rhizobacterial community and peanut growth/yield, and provide a basis for further improving peanut yield.【Method】Four treatments were set up and conducted for 9 continuous years, including control treatment (LU), black film treatment (HEI), bio-degradable film treatment (JI) and conventional film treatment (PU). Taking the rhizosphere soil of peanut at the flowering stage and mature stage in 2022 as the research object, Illumina MiSeq PE300 as the sequencing platform, and 16S rRNA gene as the target gene, effects of different types of film treatments on the structure and function of rhizobacterial community and the growth and yield of peanut were studied.【Result】Compared with LU, peanut yield was increased by -0.45%-2.34%, 2.44%-14.36% and 6.14%-24.69% in HEI, JI and PU treatment, respectively. At the flowering stage and mature stage, HEI treatment totally altered the relative abundances of 6 rhizobacterial communities at the levels of class and genus, while results of correlation analysis showed that there was no correlation between rhizobacterial community and peanut growth or yield. JI treatment totally altered 6 rhizobacterial communities at the levels of class and genus, among which, 1 class (Blastocatellia) showed positive relationship with peanut yield. PU treatment totally influenced 12 rhizobacterial communities at the levels of class and genus, of these, 1 class (Acidobacteriae) and 1 genus (Ellin6067) were correlated with peanut main stem height and lateral branch number at the flowering stage, 1 class (Clostridia) and 2 genera (Pseudomonas, Ellin6067) were correlated with peanut yield at the mature stage. The functional prediction results of rhizobacterial community showed that HEI treatment reduced the nitrogen and nitrate respiration functions at the flowering stage and mature stage; JI treatment reduced the functions of nitrate reduction, nitrogen respiration and nitrate respiration at the flowering stage, but had no significant effect on nitrogen metabolism at the mature stage; PU treatment decreased aerobic ammonia oxidation and nitrate respiration at the flowering stage, and increased these two nitrogen metabolism functions at the mature stage.【Conclusion】Black film has no significant effect on peanut yield and rhizobacterial community; Bio-degradable film can improve the structure of rhizobacterial community and yield at the mature stage, but its yield increasing effect is not stable; Conventional film possesses better improving effects than bio-degradable film in peanut yield and rhizobacterial community structure and function.

The Effect of 3-Oxyacyl ACP Reductase Gene FgOAR1 on the Growth, Development and Pathogenicity of Fusarium graminearum

GONG AnDong, LEI YinYu, WU NanNan, LIU JingRong, SONG MengGe, ZHANG YiMei, YANG Guang, YANG Peng

Scientia Agricultura Sinica. 2023, 56(24): 4854-4865. doi:10.3864/j.issn.0578-1752.2023.24.005

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【Background】Fusarium graminearum is the main pathogenic fungus which can infect wheat and result in Fusarium head blight. The infection of F. graminearum leads to huge reduction in crop quality and yield, and produces different types of mycotoxins that endanger grain production and human health. 3-oxoacyl ACP reductase (OAR1), catalyzing carboxyacyl carrier protein to oxyacyl carrier protein, which plays an important role in the fatty acid biosynthesis of F. graminearum.【Objective】To reveal the biological function of FgOAR1 in F. graminearum, the FgOAR1 deletion mutants were constructed. The phenotype, cell structure, conidia concentration, sexual reproduction and pathogenicity of mutant were analyzed and compared with wild-type strain PH-1. The results will uncover the function of FgOAR1 in the growth and pathogenic process of F. graminearum, and provide scientific evidences for the identification of novel antifungal target and control of Fusarium head blight.【Method】Wild-type strain F. graminearum PH-1 was used as material in this study. Split-Marker gene knockout technology was conducted to construct the ΔFgOAR1 mutants. The ΔFgOAR1-C reverting strain was obtained by the Gap-repair method. All strains were individually inoculated in common media (PDA, CM, YPG), and the stress selection medium containing Congo-Red, SDS, NaCl and H₂O₂, respectively. The mycelium phenotypes and fatty acid content were recorded. The strains were inoculated in CMC medium to analyze the conidia concentration. The carrot culture-medium was used to analyze the sexual reproduction of each strain. All strains were individually inoculated to the wheat coleoptile and spikelet to analyze the pathogenicity and mycotoxin content, respectively. The disease incidence of mutant strains was calculated and compared with PH-1 strain.【Result】Compared with the wild-type strain PH-1, the growth of ΔFgOAR1 mutant showed no difference in PDA, YEG and CM media. Whereas, under the pressure of 0.7 mol·L^-1 NaCl, 0.03% H₂O₂, 0.01% SDS and 300 μg·mL^-1 Congo-Red, the growth of ΔFgOAR1 mutant was significantly reduced compared with PH-1. The evidence demonstrated that FgOAR1 is related to the cell membrane and cell wall formation in F. graminearum. In CMC medium, the conidia concentration of ΔFgOAR1 mutant (8.1×10⁵ conidia/mL) was less than PH-1 (1.26×10⁶ conidia/mL) with significant difference; The pathogenicity analysis further demonstrated that the disease incidence of ΔFgOAR1 mutant was significantly lower than PH-1 in wheat coleoptile and spikelet inoculation tests.【Conclusion】FgOAR1 participates in the fatty acid biosynthesis, and plays an important role in cell membrane formation of F. graminearum. The deletion of FgOAR1 resulted in the reduced resistance to pressures, decreased conidia production and pathogenicity. Therefore, FgOAR1 is important for the growth, development and pathogenicity process of F. graminearum.

The Effect of Environmental Stress on Respiratory Rate and Expression Level of Mitochondrial Protein-Coding Genes in Cryptolestes ferrugineus

CHEH ErHu, YUAN GuoQing, SUN ShengYuan, TANG PeiAn

Scientia Agricultura Sinica. 2023, 56(24): 4866-4879. doi:10.3864/j.issn.0578-1752.2023.24.006

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【Background】Mitochondria is an important organelle in the organism, which is the primary site for cellular oxygen consumption and the production of the energy substance adenosine triphosphate (ATP), playing a significant role in the organism’s resistance to adversity. The rusty grain beetle (Cryptolestes ferrugineus) is a type of global stored grain pest, possessing extremely strong environmental adaptability.【Objective】The objective of this study is to analyze the respiratory rate of C. ferrugineus and the response of mitochondrial protein-coding genes to different environmental stresses, and to investigate the stress response of mitochondria in the adversity resistance of C. ferrugineus.【Method】Mitochondrial protein-coding genes were identified based on the mitochondrial genome data of C. ferrugineus, and corresponding real-time fluorescence quantitative PCR (RT-qPCR) primers were designed. The toxicity regression equation and LC₃₀ of C. ferrugineus to fumigants (ethyl formate), botanical insecticides (rotenone), and stored grain protectants (avermectin) were determined by using bioassay methods, and these concentrations were used for subsequent drug stress treatment on the test insects. The spatial and temporal expression patterns (different developmental stages and different tissues of larvae) of mitochondrial protein-coding genes in C. ferrugineus were analyzed by using RT-qPCR technology. Finally, the changes in the respiratory rate of C. ferrugineus under various adversity stresses such as high temperatures (35 and 40 ℃), ethyl formate, rotenone, avermectin, and starvation, as well as the expression patterns of mitochondrial protein-coding genes, were studied by using a CO₂ detector and RT-qPCR technology, respectively.【Result】Twelve mitochondrial protein-coding genes (excluding nad6) quantitative primers were designed. RT-qPCR results showed that these mitochondrial protein-coding genes had a higher expression level at the 3rd instar larval stage, and mitochondrial genes were specifically highly expressed in the Malpighian tubules of 3rd instar larvae. Moreover, under high-temperature stress, the respiratory rate of C. ferrugineus significantly increased, and the expression levels of mitochondrial protein-coding genes nad2, cytb, and cox2 increased significantly, while nad4, nad4L, cox3, and atp6 showed a significant downregulation trend. Under ethyl formate fumigation stress, the respiratory rate of C. ferrugineus significantly decreased, and all 12 mitochondrial protein-coding genes were significantly downregulated. Among them, the expression levels of nad4L and nad5 were only 3.48% and 1.91% of the control group, respectively. Under rotenone and avermectin stress, the respiratory rate of C. ferrugineus significantly decreased, and the expression levels of mitochondrial protein-coding genes, except for cox2, were significantly downregulated. Under starvation stress, the respiratory rate of C. ferrugineus significantly decreased, and as the stress duration increased, the downregulation of mitochondrial encoded gene expression levels became more pronounced.【Conclusion】The respiratory metabolism rate and mitochondrial protein-coding gene expressions of C. ferrugineus changed significantly under different environmental stresses, indicating that the mitochondria plays an important role in the adaption to high temperature, pesticides, and starvation stress in C. ferrugineus.

Relationship Between Grain Nitrogen Content and Yield Formation, Uptake and Partitioning of NPK of High-Yielding Wheat Cultivars in Drylands

XU JunFeng, ZHANG XueMei, YANG Jun, GUO ZiKang, HUANG Cui, DING YuLan, HUANG Ning, SUN RuiQing, TIAN Hui, WANG ZhaoHui, SHI Mei

Scientia Agricultura Sinica. 2023, 56(24): 4880-4894. doi:10.3864/j.issn.0578-1752.2023.24.007

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【Objective】The aim of this study was to understand the variations of grain nitrogen (N) content and its relationships with yield formation as well as uptake, transfer and partitioning of nitrogen, phosphorus, and potassium (NPK) within high-yielding wheat cultivars, which was of great significance for screening and breeding superior wheat cultivars with high yield and grain N content, and for nutrient management in green production of wheat with high yield and high quality in drylands.【Method】Field experiments were carried out, and 14 high-yielding wheat cultivars of similar yields and different grain N content were cultivated on the typical dryland area of the Loess Plateau from 2017 to 2022. Differences in grain N contents of high-yielding wheat cultivars and its relation to yields, yield components as well as N, P and K content in different organs were analyzed. Meanwhile, biomass accumulation and yield formation as well as uptake, transfer and partitioning of NPK in response to fertilization were investigated.【Result】Significant positive correlation was found between the grain N content and 1000-grain weight of wheat cultivars, and for each 1.0 g increase of the 1000-grain weight, the grain N content increased by 0.3 g·kg^-1. The grain N content showed significant differences in the tested wheat cultivars, with the mean of high-N group being 24.9 g·kg^-1, and 16% higher than that of the low-N group (21.5 g·kg^-1), respectively, while the average yields were not significantly different from each other of the two groups. The yield, biomass and spike number of high-N group exhibited higher response to N and P fertilizer application. The grain P content and straw K content of the high-N group were higher than the low-N group under different fertilization conditions, and the increases of N, P and K uptake in grain and shoot were larger than that of low-N group after N and P application. The abilities of N transfer from glumes to grains in the high-N group was greater than that in the low-N group, but the capacities of K transfer from straws to grains and glumes was lower than that in low-N cultivars. The abilities of K transfer from straws to other organs decreased significantly after N application. Application of N, P and K was conducive to increase the partitioning of K from shoots to straws of the high-N group.【Conclusion】The high-yielding wheat cultivars exhibited significant differences in the grain N content. The 1000-grain weight and grain P content of high-N cultivars were higher, its straw K content was also higher, but its capacities of K transfer from straws to grains and glumes was lower. The N, P and K uptake of the high-N group in grains and shoots increased extremely after N and P application. Therefore, to screen and breed superior wheat cultivars with high grain N content on the basis of high yield, the attention should be paid to select the cultivars with high grain 1000-grain weight as well as the strong transferring and partitioning abilities of P to grains and K to straws. The efforts should be also made to cooperate the supply of P, K and N in wheat production, with the purpose to increase the grain N content.

Characteristics of Oilseed Rape Cultivar with Different Oil Content in Nutrient Dynamitic Accumulation Rates and Utilization Efficiency

HU WenShi, LI YinShui, ZHAO ManLi, ZHANG ShanShan, GU ChiMing, DAI Jing, LI XiaoYong, YANG Lu, QIN Lu, LIAO Xing

Scientia Agricultura Sinica. 2023, 56(24): 4895-4905. doi:10.3864/j.issn.0578-1752.2023.24.008

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【Objective】To clarify the requirements of oilseed rape cultivars characterized by high-oil content for nitrogen (N), phosphorus (P), and potassium (K) nutrients, the dynamic nutrient accumulation rates of cultivars with different oil content were compared, so as to provide a theoretical basis on precise nutrient management for realizing high oil yield potential. 【Method】The field and pot experiments were carried out during 2019-2021 oilseed rape growth seasons to monitor the dynamic changes in biomass, NPK content and accumulation rates of oilseed rapes cultivars with high oil content (Zhongyouza 19, Z19) and conventional oil content control cultivars (Huayouza 12, H12, and Huayouza 62, H62). And then the differences in nutrient utilization efficiency were analyzed.【Result】The results of two-year experiments showed that the seed yield of Z19 was obviously lower than that of H62 by 8.5%-20.4%, but there was little difference in seed yield between Z19 and H12. However, the oil content of Z19 was significantly higher than those of H12 and H62 (10.1%-26.7%). By monitoring the dynamic biomass and nutrient content in the field and pot traits during the 2020/2021 growth seasons, it was claimed that the biomass of Z19 was lower than that of H62, which was higher than that of H12 at podding and mature stage with different degrees, while there was minor difference in biomass between Z19 and H12 before podding stage. The N content of Z19 was significantly lower than H12 and H62 during growth in pot experiment and after flowering stage in field experiment. And K content of Z19 was higher than H12 and H62 during growth. However, there was no consistent changes in P content between field and pot experiments among three cultivars. Consequently, the N accumulation rates of Z19 were lower than that of H62 by 19.2%-29.0% and lower than that of H12 by 9.8%-13.1% in pot experiment. The K accumulation rate of Z19 was higher than H12 by 7.4%-39.2% except for the flowering stage. Moreover, the P accumulation rate of Z19 was between those of H62 and H12, which was remarkably higher than that of H12 in the field and significantly lower than that of H62 in the pot. The N utilization efficiency and oil production efficiency of Z19 were significantly higher than those of H12 and H62. Moreover, the K utilization efficiency of Z19 was lower than that of H12 and H62, but the K oil production efficiency of Z19 was close to that of H12 and H62. There was minor difference in P utilization efficiency among 3 cultivars, but the P oil production efficiency of Z19 was significantly higher than that of H12. ¹⁵N labeling indicated that there was no obviously difference in N fertilizer use efficiency among three cultivars until flowering stage, after which N fertilizer use efficiency of Z19 was significantly lower than that of H12 and H62.【Conclusion】In summary, high-oil content cultivar, Z19, had a higher demand for K and a lower demand for N with a higher N utilization efficiency. In agricultural production, K fertilizer could be supplemented for high-oil content cultivar, and less N fertilizer could be applied in comparison to the conventional oilseed rape cultivars.

Cloning and Expression Analysis of PtMLP1 Promoter in Poncirus trifoliata

YAO LiXiao, SU Juan, GUO XingRu, LI FengLong, HE YongRui, ZOU XiuPing, CHEN ShanChun

Scientia Agricultura Sinica. 2023, 56(24): 4906-4915. doi:10.3864/j.issn.0578-1752.2023.24.009

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【Objective】Genetic transformation plays a significant role in exploring gene function and improving traits in citrus. Tissue-specific promoters is a key to regulate the expression of transgenes in particular tissues. Here, expression characteristics of the PtMLP1 promoter, isolated from the root subtractive library of Poncirus trifoliata, was thoroughly examined, which could lay a foundation for the specific expression of exogenous genes in citrus root tissue. 【Method】The complete sequence of PtMLP1 gene was cloned by PCR using DNA as a template. The physiochemical attributes, secondary and tertiary structures of PtMLP1 protein were predicted by ExPASy, PSIPRED, and SWISS-MODEL tools. Cis-acting elements in PtMLP1 promoter were predicted by PlantCARE. The expression pattern of PtMLP1 in P. trifoliata trees of diverse ages was examined by employing real-time qPCR. Furthermore, to investigate the tissue-specific expression of the PtMLP1 promoter in citrus, a pBI121-ProPtMLP1::GUS plasmid, in which GUS expression was controlled by the PtMLP1 promoter, was constructed and then introduced into P. trifoliata through Agrobacterium-mediated hypocotyl transformation. 【Result】PtMLP1 consisted of two exons and one intron, which possessed a 471 bp open reading frame encoding a protein with 156 amino acid residues. This protein had a molecular weight of 17.63 kilodaltons with an isoelectric point of 5.49 and contained a Bet v I functional domain in its primary structure. Moreover, the secondary structure of PtMLP1 contained three α-helices and seven β-folds, while its tertiary structure had a conserved hydrophobic binding site and a cyclic domain, which was rich in glycine. The PtMLP1 promoter was 1 666 bp long. Multiple root-specific expression elements, phytohormone response elements (such as the TGACG motif, P-box, and ABRE), and the TATA box and CAAT box core elements were predicted in the promoter. Additionally, the 3-terminal untranslated region of PtMLP1 was predicted to contain a poly (A) signal AATAAA. Notably, the expression of PtMLP1 was significantly higher in the roots of 1-month, 6-month, and 20-year-old P. trifoliata, with fold changes of 46.34, 74.82, and 110.25, respectively, compared with those in leaves. GUS expression analysis of pBI121-ProPtMLP1::GUS transgenic plants showed that PtMLP1 promoter exhibited specific and high expression in roots, and its expression levels were 7.76 to 124.78 times of that in the leaves. 【Conclusion】The sequences of the PtMLP1 gene and its promoter were successfully obtained, and the promoter demonstrated the ability to drive specific expression of GUS gene in citrus roots.

Effects of Intercropping with Vulpia myuros in Tea Plantation on Soil and Tea Quality Components

CHEN YiYong, LI JianLong, ZHOU Bo, WU XiaoMin, CUI YingYing, FENG ShaoMao, HU HaiTao, TANG JinChi

Scientia Agricultura Sinica. 2023, 56(24): 4916-4929. doi:10.3864/j.issn.0578-1752.2023.24.010

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【Objective】 The objective of this study was to analyze the impact of intercropping Vulpia myuros on soil nutrient composition, soil microbial community structure, and tea quality components in tea plantations, so as to provide the data support for intercropping V. myuros as a means to enhance the ecological environment and quality of tea cultivation.【Method】The experimental materials consisted of soil and fresh leaves from a tea garden that had been intercropped with V. myuros for a duration of 2 years, and the control group was a clear-ploughed tea garden. The pH, organic matter, and mineral nutrients of the topsoil in the tea garden were measured. Additionally, the population structure of soil bacteria and fungi was analyzed using 16S and ITS high-throughput sequencing techniques. The quality components of tea were determined through Agilent-7890B gas chromatography.【Result】After intercropping V. myuros in a tea garden for 2 years, the soil pH increased by 0.29, and the soil organic matter content increased by 16.46 g∙kg^-1. Additionally, the available phosphorus, available potassium, ammonium nitrogen, and nitrate nitrogen also increased to varying degrees in the tea garden soil planted with V. myuros. Notably, the available phosphorus was 5.88 times higher in the intercropped tea garden compared with the clear-cultivated tea garden. The total nitrogen content in the V. myuros plantation soil was higher than that in the clear-cultivated tea plantation, while the total phosphorus, potassium, and sodium contents were lower. Moreover, the tea garden soil planted with V. myuros had higher levels of available zinc, available iron, available copper, and cation exchange capacity. The intercropping of V. myuros in the tea garden also led to an increase in the number of bacteria and fungi in the soil. Furthermore, the relative abundance of Actinobacteria and Ascomycota associated with organic matter decomposition increased in the soil of the V. myuros tea plantation. A total of 259 metabolites were identified from the fresh leaves of the grass plantation and the clear cultivation garden. Among them, the content of 20 metabolites showed significant differences, and these different metabolites mainly included sugars, fatty acids, and catechins. The tea leaves of the V. myuros plantation had more than 2 times the contents of leucrose, methyl-β-D-glucopyranoside, lacttol alcohol, galactoglycerol, and α-lactose compared to the tea plantation. On the other hand, the content of (9Z)-octadecatrienoic acid and (9Z,12Z,15Z)-octadecatrienoic acid were significantly lower in the cultivated tea garden. Additionally, compared with the clear-ploughed tea garden the intercultivated tea plantation had significantly lower contents of (+)-galligallocatechin, galligallocatechin, and epicatechin metabolites. 【Conclusion】When V. myuros was interplanted with tea gardens, the acidity of the soil might be successfully alleviated, and the amount of organic matter and mineral nutrient components in the soil increased. Then, tea plants could more easily absorb and use soil nutrients when there were changes in the amount and community structure of soil bacteria and fungi. The structure of the microbial population and variations in soil nutrients had a significant impact on the quality of tea.

Effects of Processing Technology on the Amandin Immunoreactivity and Digestive Stability of Apricot Kernel

LONG FeiFei, ZHANG QingAn, ZHANG ZhiHua

Scientia Agricultura Sinica. 2023, 56(24): 4930-4943. doi:10.3864/j.issn.0578-1752.2023.24.011

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【Background】In recent years, allergies have become a global health concern, and the number of allergic individuals continues to rise. Nut is one of the common origins of allergies, and apricot kernels, as a common nut containing the allergenic protein amandin, have become one of the most susceptible nuts to allergies. Therefore, allergy removal of nuts has become a research hotspot. The processing of apricot kernels generally involves procedures, such as peeling, debitterizing and drying, and there are no relevant reports on whether their allergenicity will be affected during these processing. 【Objective】The aim of this study was to explore the impact of processing on the allergenicity with the allergenicity, quality and nutritional characteristics of apricot kernels as the evaluation indicators, and to optimize the processing for reducing the allergenicity of apricot kernels, thus providing the theoretical basis and technical support for the processing of low allergenic nuts products of apricot kernels.【Method】Firstly, the methods of Western blotting and ELISA were used to investigate the effects of different peeling, debitterizing and drying methods on the amandin immunoreactivity in apricot kernels. Then, the circular dichroism spectroscopy, extrinsic fluorescence spectroscopy, surface hydrophobicity and zeta potential measurements were used to study the effects of various processing methods on the structure and surface properties of amandin, and to analyze the mechanism of the immune-reactivity changes of amandin. Finally, the vitro simulation digestion experiments were conducted to investigate the digestive stability of amandin in apricot kernels before and after processing, and Western blotting analysis was conducted on the digestion products to further explore the changes in potential allergenicity of apricot kernels.【Result】In terms of allergenicity, the amandin’s immunoreactivity after being peeled by the saturated hot air and blanched decreased by 8.41% and 13.15%, respectively. After being quickly debitterized by ultrasound, the amandin’s immunoreactivity decreased by 6.79%. Blanching debitterizing had no significant effects on its immunoreactivity. After natural drying and hot air drying, the immune reactivity of the amandin significantly increased by 4.58% and 2.81%, respectively (P<0.05). Based on the impact of processing on the quality and nutritional characteristics of apricot kernels, those suitable processing methods to decrease the allergenicity of apricot kernels were optimized like the saturated hot air peeling, ultrasonic rapid debitterizing and hot air drying, and the immune reactivity of apricot kernels decreased by 15.03% under the optimal conditions. In terms of the structure of amandin, the secondary structure composition, tertiary structure, surface hydrophobicity and zeta potential have undergone certain changes during the processing. Among them, the ultrasound rapid debitterizing significantly changed the tertiary structure of amandin and enhanced its surface hydrophobicity (P<0.05), resulting in the most significant decrease in its immune reactivity. The digestive stability of the amandin after processing was significantly reduced, and the accelerated degradation rate of structures was related to the specific antigen antibody reactions in allergenic proteins, leading to a further decrease in the potential allergenicity of apricot kernels.【Conclusion】Different processing steps could affect the allergenicity of apricot kernels by changing the structure of amandin, i.e. the reasonable processing methods could be used to reduce the allergenicity of apricot kernels.

LNC721 Targeted Regulation MMP9 Affects Bovineskeletal Muscle Satellite Cell Proliferation and Differentiation

GUO YunPeng, TAN HaoYun, GUO Hong, FU MengYun, LI Xin, HU DeBao, ZHANG LinLin, DING XiangBin, GUO YiWen

Scientia Agricultura Sinica. 2023, 56(24): 4944-4955. doi:10.3864/j.issn.0578-1752.2023.24.012

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【Background】The muscular system is an important basis for maintaining the survival and growth of the animal body. In the mammalian muscle, nearly half of the muscle is skeletal muscle, skeletal muscle through cell multiplication to migration fusion, and gradually formed a mature muscle bundle attached to the bone with contractile ability. In the animal body, skeletal muscle is not only involved in animal growth, but also in physiological activities, such as respiration and metabolism. At present, many studies have shown that lncRNA had the effect of regulating muscle growth and development, and was a key factor affecting skeletal muscle function and diseases. However, due to the complex mechanism of action of lncRNA, the variety of methods, and the very low conservation type between species, the relationship between lncRNAs of different species is not large, and most of the studies exist in organisms such as humans and mice, and there are few studies on the effect on bovine muscle growth. In recent years, it has been discovered that lncRNAs interact with certain target genes to regulate the process of muscle cell genesis. In the early stage of this experiment, the high-throughput sequencing was performed by collecting bovine muscles of different tissues of different months, the lncRNA with high expression difference was obtained, and the mechanism of its regulation of the myogenic process was studied. 【Objective】This study aimed to explore the interaction between long non-coding RNA lnc721 and its target gene MMP9, and the effects of MMP9 on the growth and development of bovine skeletal muscle cells, in order to provide a reference for the study of the regulatory mechanism of bovine skeletal muscle development.【Method】 The previous experiments showed that interference with lnc721 had a positive regulatory effect on the proliferation of bovine skeletal muscle satellite cells and negatively regulated its differentiation. Three groups of interfering lnc721 bovine skeletal muscle satellite cells and three control groups were set up to sequence transcriptome using NGS technology in the differentiation stage of bovine skeletal muscle satellite cells, in order to obtain the lnc721 differential target gene and to further study the regulatory pathway of lnc721 on bovine skeletal muscle development. According to the screening results and the verification results of qRT-PCR, MMP9 was selected as the target gene of lnc721, and the binding ability of lnc721 and MMP9 was predicted through the CatRAPID website. The interference sequences of lnc721 and MMP9 were designed and synthesized, transfected into bovine skeletal muscle satellite cells, and the effect of lnc721 on MMP9 expression was down-regulated by qRT-PCR and Western blot technology at the mRNA level and protein level. After down-regulation of MMP9, qRT-PCR, Western blot and EdU were used to detect the expression of proliferation marker factors Ki67 and Pax7 and differentiation marker factors MyHC and MyOG, so as to reflect the effect of down-regulation of MMP9 on the growth and development of bovine skeletal muscle satellite cells. 【Result】 MMP9 was identified as a target gene for lnc721 to regulate the interaction of bovine skeletal muscle satellite cells. They were found to interact and bind to each other by RIP. After interfering with lnc721, qRT-PCR analysis showed that down-regulation of lnc721 significantly inhibited MMP9 expression (P<0.01) during the proliferative phase, while it significantly promoted MMP9 expression (P<0.01) during the differentiation phase. Downregulation of MMP9 resulted in a highly significant upregulation of Ki67 mRNA level expression in proliferating cells (P<0.01) and the Pax7 protein expression (P ˂0.05). As also, it could significant increase the positive cell rate of EdU labled cells. At the stage of cell differentiation, the downregulation of MMP9 could inhibit muscle myotube formation. On the other hand, the mRNA and protein expressions of MyHC were significantly decreased (P<0.01); MyoG protein expression was significantly down-regulated (P<0.05). 【Conclusion】 lnc721 could bind to MMP9. Interfering with lnc721 was significantly inhibited MMP9 expression during the proliferative phase of cells, while promoting MMP9 expression during the differentiation phase. MMP9 inhibition could promoted cell proliferation and inhibited differentiation. This study demonstrated that lnc721 targeting MMP9 regulated the development of bovine skeletal muscle satellite cells.

Screening and Identification of Nanobodies Against Novel Goose Astrovirus ORF2 Protein

WANG Dan, JI YanHong, LIANG ShiRui, YANG Jie, ZHU QiYun

Scientia Agricultura Sinica. 2023, 56(24): 4956-4966. doi:10.3864/j.issn.0578-1752.2023.24.013

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【Objective】 The gosling gout disease caused by the novel goose astrovirus (nGAstV) has brought significant economic losses to the goose industry. In this study, a nanobody phage display library for nGAstV was constructed to obtain the specific nanobodies (Nbs) that recognize the ORF2 protein of nGAstV, which could pave a way for the establishment of antibody-based detection methods and the study on the structure and function of nGAstV ORF2 protein. 【Method】 The proliferated nGAstV in LMH cells was purified by sucrose gradient centrifugation. nGAstV was identified by RT-PCR and the virus titer was determined by cytopathic effect. The two-year-old alpacas were immunized with purified nGAstV inactivated by 0.6% formaldehyde solution. For the first immunization, inactivated nGAstV was emulsified with an equal volume of Freund's complete adjuvant. For the second to fifth immunization, inactivated nGAstV was emulsified with an equal volume of Freund's incomplete adjuvant. The immunization was performed every two weeks with a dose of 50 μg. And the titer of IgG against nGAstV in alpaca serum collected at 14 days post the fifth immunization was determined by indirect ELISA. When the IgG titer reached the standard for constructing a library, the alpaca peripheral blood lymphocytes (PBL) were isolated. The total RNA of PBL was extracted and reverse-transcribed into cDNA. The variable region gene of the heavy chain was amplified by nested PCR. It was constructed into pComb phage vector and combined with phage display technology to construct nGAstV Nb phage display library. The capacity of the library was calculated and its diversity was analyzed. nGAstV was used as a target antigen for three rounds of enrichment and panning to obtain recombinant phage Nb positive clones. The positive clones were then cloned into pcDNA3.1-Fc eukaryotic expression vectors followed by sequencing analysis. The plasmids with different sequences were transfected into HEK-293F cells, and the expression level was identified by SDS-PAGE. The nGAstV was used as a target antigen to test the specificity and binding activity of the expressed Nb by ELISA and Western blot. The affinity of Nb was verified using indirect ELISA using nGAstV ORF2 protein as the target antigen and to screen Nbs with better biological activity. 【Result】 The results of RT-PCR showed that nGAstV was ready to be proliferated in LMH cells. The titer of nGAstV virus was 4.38 Log₁₀TCID₅₀/mL by calculated by the Reed-Muench method. The titer of nGAstV antibodies in alpaca serum reached over 1:64 000 after five immunizations. The VHH gene was amplified by nested PCR and a phage display library of nGAstV Nb with a library capacity of 3.8×10⁸ CFU/mL was successfully constructed. Phylogenetic tree analysis showed that the phage Nb library had an excellent diversity. 39 phage positive clones were acquired, which reacted with nGAstV post three rounds of enrichment and panning, including 25 Nbs with different sequences. The results of SDS-PAGE identification showed that a total of 10 Nbs were successfully expressed. Among them, 8 Nbs that reacted explicitly with nGAstV ORF2 protein were further confirmed by ELISA and western blot, among which one Nb showed the best biological activity. 【Conclusion】 In this study, Nbs that reacted specifically with nGAstV ORF2 protein were screened for the first time, which provided materials for basic research and developing nGAstV detection methods.

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