Search

Thoracolumbar vertebra (TLV) and rib primordium (RP) development is a common evolutionary feature across vertebrates, although whole-organism analysis of TLV and RP gene expression dynamics has been lacking. Here we investigated the single-cell transcriptomic landscape of thoracic vertebra (TV), lumbar vertebra (LV), and RP cells from a pig embryo at 27 days post-fertilization (dpf) and identified six cell types with distinct gene-expression signatures. In-depth dissection of the gene-expression dynamics and RNA velocity revealed a coupled process of osteogenesis and angiogenesis during TLV and rib development. Further analysis of cell-type-specific and strand-specific expression uncovered the extremely high levels of Hoxa10 3'-untranslated region (UTR) sequence specific to osteoblast of LV cells, which may function as anti-Hoxa10-antisense by counteracting the Hoxa10-antisense effect to determine TLV transition. Thus, this work provides a valuable resource for understanding embryonic osteogenesis and angiogenesis underlying vertebrate TLV and RP development at the cell-type-specific resolution, which serves as a comprehensive view on the transcriptional profile of animal embryo development.

Aerobic denitrifiers have the potential to reduce nitrate in polluted water under aerobic conditions. A salt-tolerant aerobic denitrifier was newly isolated and identified as Vibrio spp. AD2 from a marine recirculating aquaculture system, in which denitrification performance was investigated via single-factor experiment, Box–Behnken experiment, and nitrogen balance analysis. Nitrate reductase genes were identified by polymerase chain reaction. Results showed that strain AD2 removed 98.9% of nitrate-nitrogen (NO3−-N) with an initial concentration about 100 mg·L−1 in 48 h without nitrite-nitrogen (NO2−-N) accumulation. Nitrogen balance indicated that approximately 17.5% of the initial NO3−-N was utilized for bacteria synthesis themselves, 4.02% was converted to organic nitrogen, 39.8% was converted to nitrous oxide (N2O), and 31.1% was converted to nitrogen (N2). Response surface methodology experiment showed that the maximum removal of total nitrogen (TN) occurred under the condition of C/N ratio 11.5, shaking speed 127.9 rpm, and temperature 30.8 °C. Sequence amplification indicated that the denitrification genes, napA and nirS, were present in strain AD2. These results indicated that the strain AD2 has potential applications for removing NO3−-N from high-salinity (3%) wastewater.

Blastocyst complementation by pluripotent stem cell (PSC) injection is believed to be the most promising method to generate xenogeneic organs. However, ethical issues prevent the study of human chimeras in the late embryonic stage of development. Primate embryonic stem cells (ESCs), which have similar pluripotency to human ESCs, are a good model for studying interspecies chimerism and organ generation. However, whether primate ESCs can be used in xenogenous grafts remains unclear. In this study, we evaluated the chimeric ability of cynomolgus monkey (Macaca fascicularis) ESCs (cmESCs) in pigs, which are excellent hosts because of their many similarities to humans. We report an optimized culture medium that enhanced the anti-apoptotic ability of cmESCs and improved the development of chimeric embryos, in which domesticated cmESCs (D-ESCs) injected into pig blastocysts differentiated into cells of all three germ layers. In addition, we obtained two neonatal interspecies chimeras, in which we observed tissue-specific D-ESC differentiation. Taken together, the results demonstrate the capability of D-ESCs to integrate and differentiate into functional cells in a porcine model, with a chimeric ratio of 0.001–0.0001 in different neonate tissues. We believe this work will facilitate future developments in xenogeneic organogenesis, bringing us one step closer to producing tissue-specific functional cells and organs in a large animal model through interspecies blastocyst complementation. Electronic supplementary material The online version of this article (10.1007/s13238-019-00676-8) contains supplementary material, which is available to authorized users.

Thoracolumbar vertebra (TLV) and rib primordium (RP) development is a common evolutionary feature across vertebrates although whole-organism analysis of TLV and RP gene expression dynamics has been lacking. Here we investigated the single-cell transcriptomic landscape of thoracic vertebra (TV), lumbar vertebra (LV), and RP cells from a pig embryo at 27 days post-fertilization (dpf) and identified six cell types with distinct gene-expression signatures. In-depth dissection of the gene-expression dynamics and RNA velocity revealed a coupled process of osteogenesis and angiogenesis during TLV and rib development. Further analysis of cell-type-specific and strand-specific expression uncovered the extremely high levels of HOXA10 3’-UTR sequence specific to osteoblast of LV cells, which may function as anti-HOXA10-antisense by counteracting the HOXA10-antisense effect to determine TLV transition. Thus, this work provides a valuable resource for understanding embryonic osteogenesis and angiogenesis underlying vertebrate TLV and RP development at the cell-type-specific resolution, which serves as a comprehensive view on the transcriptional profile of animal embryo development.

A variety of tumors are insensitive to immune checkpoint blockade (ICB) therapy. We propose that ICB therapy alone is insufficient to fully reactivate antitumor T cells, while effective mobilization of antigen-presenting cells (APCs) to assist adaptive immune cell activation can lead to potent antitumor effects with broad responsiveness. The Toll-like receptor 7 (TLR7) agonist SZU-101 we developed can induce the innate immune response against tumors and increase the immunogenicity of tumors. Interestingly, SZU-101-induced upregulation of programmed death ligand 1 (PD-L1) expression in tumor tissues can further enhance the response rate of the PD-L1 antibody. In addition, PD-L1 nanobodies have better solid tumor penetration ability, and because of this ability, they can be used to precisely deliver SZU-101 to tumor tissues. Therefore, a PD-L1 and TLR7 dual-targeting nanobody-drug conjugate (NDC), a novel drug molecule, was developed. We found that TLR7 agonists and PD-L1 nanobodies act synergistically and that NDC treatment reshapes the tumor immune microenvironment, activates both innate and adaptive immune cells, and exerts antitumor effects in both “hot” and “cold” tumors primarily through CD8+ T cells and natural killer (NK) cells. Our data show that a PD-L1 and TLR7 dual-targeting NDC can exhibit potent antitumor efficacy by orchestrating innate and adaptive immune responses and shows good prospects for clinical development.One Sentence SummaryBased on results showing that TLR7 agonists and PD-L1 nanobodies exert synergistic antitumor effects, a PD-L1 and TLR7 dual-targeting nanobody-drug conjugate that we developed shows good prospects for clinical development because it can orchestrate innate and adaptive immune responses, reshape the tumor immune microenvironment, and exert potent antitumor effects against both “hot” and “cold” tumors.

Two kinds of thermoresponsive 2-hydroxy-3-alkoxypropyl hydroxyethyl celluloses (HAPEC) were prepared by grafting butyl and isopropyl glycidyl ethers onto hydroxyethyl celluloses (HEC). The HAPEC was characterized by 1H NMR, 13C NMR, and 2D HSQC NMR. The lower critical solution temperature (LCST) of HAPEC can be tuned by changing the molar substitution (MS). The LCST decreased with the increasing MS of the alkyl chains. The HAPEC concentration, salt concentration, and organic solvent concentration had a marked influence on LCST. In addition, the differences of thermoresponsive properties between the two kinds of HAPECs were investigated. 2-Hydroxy-3-butoxypropyl hydroxyethyl cellulose (HBPEC), which has longer hydrophobic side chains, demonstrated a lower LCST when both HBPEC and 2-hydroxy-3-isopropoxypropyl hydroxyethyl cellulose (HIPEC) possessed similar MS values. HBPEC, which has longer hydrophobic side chains, exhibited thermoresponsive flocculation behavior, and the critical flocculation temperature (CFT) was adjusted in the range from 27.3 to 51.2 °C by changing the molar substitution.

This study investigated an Iron–carbon (Fe-C) micro-electrolysis method to enhance nitrogen removal of Sesuvium portulacastrum constructed wetlands (CWs) when treating mariculture effluents. The main objective was to investigate the effects of Fe-C on nitrogen purification performance and microbial characteristics of Sesuvium portulacastrum CWs. Results showed that the presence of Fe-C and Sesuvium portulacastrum could improve nitrogen removal efficiency by 20–30% and 15–30%, respectively. CWs with 33% v/v Fe-C addition performed well on nitrogen removal: TAN, 41.49 ± 13.64%; NO2–-N, 13.32%; NO3–-N, 60.02 ± 6.17%; TIN, 63.40 ± 12.11%. Microbial analysis revealed that Fe-C altered the microbial communities, and improved the abundance of denitrification related genera. Based on microbial enzyme activities and genes abundance, the anammox and denitrification processes were promoted by Fe-C in CWs. These findings indicate that Sesuvium portulacastrum CWs with 33% v/v Fe-C represents an effective nitrogen removal for mariculture wastewater with insufficient carbon source.

Objectives Immunodeficient mice injected with human cancer cell lines have been used for human oncology studies and anti‐cancer drug trials for several decades. However, rodents are not ideal species for modelling human cancer because rodents are physiologically dissimilar to humans. Therefore, anti‐tumour drugs tested effective in rodents have a failure rate of 90% or higher in phase III clinical trials. Pigs are similar to humans in size, anatomy, physiology and drug metabolism rate, rendering them a desirable pre‐clinical animal model for assessing anti‐cancer drugs. However, xenogeneic immune rejection is a major barrier to the use of pigs as hosts for human tumours. Interleukin (IL)‐2 receptor γ (IL2RG), a common signalling subunit for multiple immune cytokines including IL‐2, IL‐4, IL‐7, IL‐9, IL‐15 and IL‐21, is required for proper lymphoid development. Materials and Methods IL2RG −/Y pigs were generated by CRISPR/Cas9 technology, and examined for immunodeficiency and ability to support human oncogenesis. Results Compared to age‐matched wild‐type pigs, IL2RG −/Y pigs exhibited a severely impaired immune system as shown by lymphopenia, lymphoid organ atrophy, poor immunoglobulin function, and T‐ and NK‐cell deficiency. Human melanoma Mel888 cells generated tumours in IL2RG −/Y pigs but not in wild‐type littermates. The human tumours grew faster in IL2RG −/Y pigs than in nude mice. Conclusions Our results indicate that these pigs are promising hosts for modelling human cancer in vivo, which may aid in the discovery and development of anti‐cancer drugs.

Background Xenogeneic organ transplantation has been proposed as a potential approach to fundamentally solve organ shortage problem. Xenogeneic immune responses across species is one of the major obstacles for clinic application of xeno-organ transplantation. The generation of glycoprotein galactosyltransferase α 1, 3 (GGTA1) knockout pigs has greatly contributed to the reduction of hyperacute xenograft rejection. However, severe xenograft rejection can still be induced by xenoimmune responses to the porcine major histocompatibility complex antigens swine leukocyte antigen class I and class II. Methods We simultaneously depleted GGTA1, β2-microglobulin (β2M), and major histocompatibility complex class II transactivator (CIITA) genes using clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins technology in Bamma pig fibroblast cells, which were further used to generate GGTA1β2MCIITA triple knockout (GBC-3KO) pigs by nuclear transfer. Results The genotype of GBC-3KO pigs was confirmed by polymerase chain reaction and Sanger sequencing, and the loss of expression of α-1,3-galactose, SLA-I, and SLA-II was demonstrated by flow cytometric analysis using fluorescent-conjugated lectin from bandeiraea simplicifolia, anti-β2-microglobulin, and swine leukocyte antigen class II DR antibodies. Furthermore, mixed lymphocyte reaction assay revealed that peripheral blood mononuclear cells from GBC-3KO pigs were significantly less effective than (WT) pig peripheral blood mononuclear cells in inducing human CD3CD4 and CD3CD8 T-cell activation and proliferation. In addition, GBC-3KO pig skin grafts showed a significantly prolonged survival in immunocompetent C57BL/6 mice, when compared with wild-type pig skin grafts. Conclusions Taken together, these results demonstrate that elimination of GGTA1, β2M, and CIITA genes in pigs can effectively alleviate xenogeneic immune responses and prolong pig organ survival in xenogenesis. We believe that this work will facilitate future research in xenotransplantation.

Aerobic denitrifiers have the potential to reduce nitrate in polluted water under aerobic conditions. A salt-tolerant aerobic denitrifier was newly isolated and identified as Vibrio spp. AD2 from a marine recirculating aquaculture system, in which denitrification performance was investigated via single-factor experiment, Box-Behnken experiment, and nitrogen balance analysis. Nitrate reductase genes were identified by polymerase chain reaction. Results showed that strain AD2 removed 98.9% of nitrate-nitrogen (NO

Insulin-like growth factor 2 (IGF2) is an important growth factor, which promotes growth and development in mammals during fetal and postnatal stages. Using CRISPR-Cas9 system, we generated multiple founder pigs containing 12 different mutant alleles around a regulatory element within the intron 3 of IGF2 gene. Crossing two male founders passed four mutant alleles onto F1 generation, and these mutations abolished repressor ZBED6 binding and rendered this regulatory element nonfunctional. Both founders and F1 animals showed significantly faster growth, without affecting meat quality. These results indicated that editing IGF2 intron 3-3072 site using CRISPR-Cas9 technology improved meat production in Bama pigs. This is the first demonstration that editing non-coding region can improve economic traits in livestock.

A novel bacteria with heterotrophic nitrification and aerobic denitrification ability was obtained from a membrane bioreactor (MBR) and identified as Acinetobacter sp. TSH1. The nitrogen removal characteristics, nitrogen balance analysis, kinetic characteristics, and enhanced biological treatment in MBR of the novel isolated strain TSH1 were determined. Results showed that strain TSH1 could remove approximately 96.6% of NH4+-N, 82.9% of NO2−-N and 98.7% of NO3−-N in 24 h, and the corresponding maximum removal rates were 3.64 mg-N/(L·h), 1.77 mg-N/(L·h) and 3.94 mg-N/(L·h). The nitrogen balance analysis indicated that most of NH4+-N (62.6%) and NO3−-N (71.9%) were transformed to gaseous nitrogen. The kinetic experiments showed that strain TSH1 had a high Km of 151.64 mg-NH4+-N/L and 203.25 mg-NO3--N/L. The enhanced biological treatment of synthetic wastewater in MBR showed that the strain TSH1 can significantly improve the nitrogen removal efficiency.

The study aimed to isolate a novel strain with heterotrophic nitrification and aerobic denitrification ability and evaluate the nitrogen removal characteristics. Results showed that Ochrobactrum anthropi HND19 could remove approximately 98.6% of NH4+-N (104.3 mg·L-1) and 97.6% of NO3−-N (98.6 mg·L-1), and the removal rates achieved 4.28 and 4.01 mg-N/(L·h) by heterotrophic nitrification and aerobic denitrification. The optimal incubate conditions of strain HND19 were 120 rpm (shaking speed), 5 ‰ (salinity), 30 °C (temperature), 7.5 (C/N ratio) with sodium acetate as carbon resource. And the removal efficiency of the total nitrogen (TN) realized 73.4% under the optimal conditions. Functional genes (hao, napA, nirK, norB, and nosZ) involved in the nitrogen removal processes were successfully amplified from strain HND19. These findings indicate that the strain HND19 possesses great application feasibility in treating wastewater with high-intensity nitrogen.

Background: The generation of glycoprotein galactosyltransferase alpha 1, 3 (GGTA1) knockout pigs has greatly contributed to the reduction of hyperacute xenograft rejection. However, severe xenograft rejection can still be induced by xenoimmune responses to the porcine major histocompatibility complex (MHC) antigens SLA-I and SLA-II. Methods: We simultaneously depleted GGTA1, β 2M, and CIITA genes using CRISPR-Cas9 technology to generate GGTA1-/-β2M-/-CIITA-/- triple knockout (GBC-3KO) pigs. Mixed lymphocyte reaction (MLR) and BrdU incorporating assays were utilized to quantify the potency of pig cells to stimulate human T cell responses. Skin grafts were determined the immunogenicity of pig tissues in xenogeneic hosts in vivo. Findings: The genotype of GBC-3KO pigs was confirmed by PCR and Sanger sequencing, and the loss of expression of α-Gal, SLA-I, and SLA-II was demonstrated by flow cytometric analysis. Mixed lymphocyte reaction (MLR) assay revealed that splenocytes from GBC-3KO pigs were significantly less effective than WT pig splenocytes in inducing human CD3+CD4+ and CD3+CD8+ T cell activation (measured by CD25 expression) and proliferation. Furthermore, GBC-3KO pig skin grafts showed a significantly prolonged survival (with a median survival time (MST) of 16 days) in immunocompetent C57BL/6 mice, when compared to WT pig skin grafts (MST = 13.5 days). Interpretation: These results demonstrate that elimination of GGTA1, β2M, and CIITA genes in pigs can effectively alleviate xenogeneic immune responses. Funding Statement: This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030000 and XDA16030303), the National Key Research and Development Program (2017YFA0104400, 2016YFA0100202 and 2017YFA0104402), the National Natural Science Foundation of China (31621004, 81570145, 81870091), and the Key Research Projects of the Frontier Science of the Chinese Academy of Sciences (QYZDB-SSW-SMC022). Declaration of Interests: The authors stated: "None." Ethics Approval Statement: Bama miniature pigs were raised and cloned at the Beijing Farm Animal Research Center. Human blood was obtained from healthy volunteers. Protocols related to the use of animals and human tissues were approved by the Institutional Review Board and Institutional Animal Care and Use Committee of the First Hospital of Jilin University and the Institute of Zoology of Chinese Academy of Sciences. All the experiments were performed in accordance with the protocols.

Significance To investigate the epigenetic mechanism of pregnancy failure in mammals, we exploited the high rate of fetal loss in pig induced pluripotent stem cell (iPSC) nuclear transfer. We generated methylomes of pig iPSCs and associated nuclear transfer embryos from reciprocal crosses between two distinct pig breeds. Our methylome analysis revealed that misregulation of RTL1 as the principal basis of pregnancy failure using pig iPSCs. Remarkably, RTL1 has broad fertility implications across mouse, rat, pig, cattle, and human from nuclear transfer cloning, tetraploid complementation, and artificial insemination, to natural fertilization. In all of these procedures, low RTL1 expression consistently corresponds to pregnancy failures., Substantial rates of fetal loss plague all in vitro procedures involving embryo manipulations, including human-assisted reproduction, and are especially problematic for mammalian cloning where over 90% of reconstructed nuclear transfer embryos are typically lost during pregnancy. However, the epigenetic mechanism of these pregnancy failures has not been well described. Here we performed methylome and transcriptome analyses of pig induced pluripotent stem cells and associated cloned embryos, and revealed that aberrant silencing of imprinted genes, in particular the retrotransposon-derived RTL1 gene, is the principal epigenetic cause of pregnancy failure. Remarkably, restoration of RTL1 expression in pig induced pluripotent stem cells rescued fetal loss. Furthermore, in other mammals, including humans, low RTL1 levels appear to be the main epigenetic cause of pregnancy failure.

Aerobic denitrification microbes have great potential to solve the problem of NO3&minus, N accumulation in industrialized recirculating aquaculture systems (RASs). A novel salt-tolerant aerobic denitrifier was isolated from a marine recirculating aquaculture system (RAS) and identified as Halomonas alkaliphile HRL-9. Its aerobic denitrification performance in different dissolved oxygen concentrations, temperatures, and C/N ratios was studied. Investigations into nitrogen balance and nitrate reductase genes (napA and narG) were also carried out. The results showed that the optimal conditions for nitrate removal were temperature of 30 &deg, C, a shaking speed of 150 rpm, and a C/N ratio of 10. For nitrate nitrogen (NO3&minus, N) (initial concentration 101.8 mg&middot, L&minus, 1), the sole nitrogen source of the growth of HRL-9, the maximum NO3&minus, N removal efficiency reached 98.0% after 24 h and the maximum total nitrogen removal efficiency was 77.3% after 48 h. Nitrogen balance analysis showed that 21.7% of NO3&minus, N was converted into intracellular nitrogen, 3.3% of NO3&minus, N was converted into other nitrification products (i.e., nitrous nitrogen, ammonium nitrogen, and organic nitrogen), and 74.5% of NO3&minus, N might be converted to gaseous products. The identification of functional genes confirmed the existence of the napA gene in strain HRL-9, but no narG gene was found. These results confirm that the aerobic denitrification strain, Halomonas alkaliphile HRL-9, which has excellent aerobic denitrification abilities, can also help us understand the microbiological mechanism and transformation pathway of aerobic denitrification in RASs.

With the advancements of stem cells and regenerative medicine, interspecies chimera has become a hot topic and will pave a new way of providing donor sources in organ transplantation. However, the interspecies chimera is confronted with a number of scientific questions and technical obstacles, including selections of appropriate embryonic stage and appropriate culture medium; those factors will deeply influence the developmental balance between donor cells and receptor embryos. Due to its relatively rapid reproductive cycle and similar organ size to human's, porcine is a very potential donor candidate to study these questions. To compare the development and chimeric efficiency of interspecies embryos, we tested and evaluated three different culture systems, PZM-3 (Porcine zygotic medium), culture medium for iPSCs (N2B27) and 3.5 h of N2B27 before PZM-3 (N2B27(3.5 h)), and two different embryonic stages, 8-cell and blastocyst in mouse-porcine chimeric embryos using parthenogenetically activated porcine embryos and mouse induced pluripotent stem cells (miPS). The results showed that, PZM-3 was beneficial for both development of chimeric embryos and miPSCs proliferation in porcine embryos in the 8-cell injection group. After early blastocyst injection, the chimeric efficiency did not appear significantly different among the three culture systems but was lower than 8-cell injection. In summary, the results suggest that 8-cell injection and PZM-3 culture medium are more beneficial to the in vitro development and chimeric efficiency of mouse-porcine chimeric embryos.近年来，随着干细胞分化与再生医学研究的不断深入，异种嵌合已成为当前干细胞和再生医学领域的热点问题，并有望为未来解决器官移植供体来源严重短缺等再生医学难题开辟新的方向。异种嵌合以及异种器官再造过程中面临众多科学问题和技术难题，而异种嵌合过程中嵌合胚胎时期的选择，后续培养液的选择以及这些环节所造成的供体细胞与受体胚胎之间的发育平衡成为建立异种器官再造的第一个科学问题。猪由于具有与人类器官大小相似、繁殖快等特点，成为异种嵌合最适合的潜在研究对象。为了提高鼠-猪异种嵌合胚胎中小鼠供体细胞——诱导多潜能干细胞 (Induced pluripotent stem cells, iPSCs) 的存活率和增殖率，我们尝试以iPSCs培养液 (N2B27) 以及N2B27→PZM-3梯度更换的培养液 (N2B27(3.5 h)) 作为研究异种嵌合胚胎体外发育培养的对象，并与猪胚胎培养液 (PZM-3，Porcine zygotic medium)体系下发育进行比较，从而评价了这3种培养液在8-细胞和囊胚期注射后，对嵌合胚胎后续发育的影响及嵌合情况。结果显示，8-细胞期注射后，PZM-3不仅对嵌合胚胎的后续发育较为有利，更有利于小鼠iPS嵌合到猪胚胎中；囊胚期注射后3种培养体系下GFP阳性嵌合率差异不显著，但其嵌合率显著低于8-细胞期嵌合率。结果表明，PZM-3培养体系更有利于鼠-猪异种嵌合胚胎的体外发育，对8-细胞期胚胎进行嵌合操作有益于提高鼠-猪异种嵌合后胚胎的嵌合率。.

Gravel-sand mulch has been used for centuries to conserve water in the Loess Plateau of northwestern China. In this study, we assessed the influence of long-term (1996–2012) gravel-sand mulching of cultivated soils on total organic carbon (TOC), light fraction organic carbon (LFOC), microbial biomass carbon (MBC), total organic nitrogen (TON), particulate organic carbon (POC), mineral-associated organic carbon (MOC), permanganate-oxidizable carbon (KMnO4-C), and non-KMnO4-C at 0–60 cm depths. Mulching durations were 7, 11 and 16 years, with a non-mulched control. Compared to the control, there was no significant and consistently positive effect of the mulch on TOC, POC, MOC, KMnO4-C and non-KMnO4-C before 11 years of mulching, and these organic C fractions generally decreased significantly by 16 years. LFOC, TON and MBC to at a 0–20 cm depth increased with increasing mulching duration until 11 years, and then these fractions decreased significantly between 11 and 16 years, reaching values comparable to or lower than those in the control. KMnO4-C was most strongly correlated with the labile soil C fractions. Our findings suggest that although gravel-sand mulch may conserve soil moisture, it may also lead to long-term decreases in labile soil organic C fractions and total organic N in the study area. The addition of manure or composted manure would be a good choice to reverse the soil deterioration that occurs after 11 years by increasing the inputs of organic matter.

Summary In the Loess Plateau of northwestern China, a system for dry farming has evolved based on the employ of gravel mulch. A couple of lab experiments were conducted to study the influences of mulch stratum thickness and gravel grain size on water vapor flow, with a focus on resistance to evaporation in gravel mulch stratum. In Experiment 1, six treatments included mulching with gravel of different thickness (2 cm, 4 cm, 6 cm, 8 cm and 10 cm) plus no mulching (control) were studied. In Experiment 2, the 10 cm thick mulch layer consisted of different grain size gravel [2–5 (A), 5–20 (B), 20–40 (C), 40–60 (D) and 60–80 (E) mm], plus three mixture treatments. Compared to bare soil, mulched soils had significantly lower accumulated evaporation, and gravel mulch significantly increased resistance to evaporation. The aerodynamic resistance to evaporation in bare soil is higher than that in mulched treatments and the relationship between equivalent grain size and aerodynamic resistance in mulched surface can be described by a line function. The relationships between mulch resistance and mulch stratum thickness or grain size of gravel, were represented by logistic curves. The findings showed that equivalent grain size and specific surface area of gravel were sensitive indicators of mulch resistance. Based on the results of laboratory experiments, we put forward a new calculated model of mulch resistance, but further research is needed for verification and exact parameterization of this model under field conditions.