Baculoviruses have been considered as the powerful vectors to express the exogenous gene. And the representative vectors in baculovirus expression vector system is Autographa californica multinucleocapsid nucleopolyhedrovirus （AcMNPV） and Bombyx mori nucleopolyhedrovirus （BmNPV）. The AcMNPV expression system has been widely applied in American and European countries. However, the BmNPV expression reaches a higher level over other systems, because BmNPV can infect silkworm larva or pupa. Moreover, silkworm is pretty normal in China, with lower cost and mature breeding technology, thus it is really popular to use the silkworm as a “biofactory” to produce recombinant protein. The BmNPV genome sequenced in 1999 was 128 413 nucleotides long with a G+C content of 40% and contained about 136 open reading frames (ORFs) encoding predicted proteins of over 60 amino acids. The gene of BmNPV orf98 is found in all Group Ⅰ and the most of Group Ⅱ genomes. It is not a highly conserved gene, as the deletion of this gene in BmNPV, it has no apparent effect on infectivity. The function of BmNPV orf98 has not been reported until now. In order to study the specific function of BmNPV orf98 gene, a BmNPV orf98 knockout bacmid by λRed recombination was constructed, naming Bm98-ko-Bacmid. Additionally, Bm98-re-Bacmid was constructed by Bac-to-Bac system. BmN cells were infected with three kinds of virus DNA from wild-type bacmid (wtBacmid), Bm98-ko-Bacmid and Bm98-re-Bacmid, and the cells were respectively collected in 12 h, 24 h, 48 h and 72 h phases, then virus titer (50% tissue culture infective dose, TCID50) was determinated and virus proliferation curve was drawn, and total DNA was extracted using a eukaryotic DNA extraction kit. After DpnⅠ enzyme digestion overnight, the effects of lacking BmNPV orf98 gene on virus replication and transcription were analyzed by fluorescence quantitative polymerase chain reaction (PCR). The results showed that the knockout bacmid was able to produce viral progeny after transfecting the DNA of Bm98 -ko-Bacmid into BmN cells, but the number of viral progeny reduced significantly （P<0.05）; meanwhile, the virus infection level of repair was recovered which was similar with that of wild-type virus, indicating that the virus infection level in the incidence of BmN cells could be delayed after the deletion of BmNPV orf98. Assembly of virus was observed by transmission electron microscope in 48 h phase. The results indicated that, after 48 h of transfecting host cell, wtBacmid and Bm98-re-Bacmid viruses produced a large number of virus-packed capsule membrane except the BmNPV orf98 knockout virus. The BmNPV orf98 deletion didn’t show significantly effects on viral DNA replication, suggesting that it was not essential for viral replication; however, the transcription of early gene lef3, late gene vp39 and very late gene p10 decreased obviously （P<0.05）, because of lack of BmNPV orf98 gene. All the above results show that BmNPV orf98 gene is non-essential for viral replication, but it can significantly affect viral progeny and assembly, and lack of the gene will lead to significant decline of the transcription level of early gene lef3, late gene vp39 and very late gene p10.

The application of controlled atmosphere (CA), is an effective, safe, and residue-free alternative compared with chemical fumigants and protectants against pests and mites, and using of carbon dioxide (CO2) has received considerable attention in disinfesting stored foodstuffs, particularly durable products. There are reports about the toxic effects of CO2-enriched atmospheres on several insect pests of stored products, but the data on the toxicity of CO2 against Oryzaephilus surinamensis Linne are limited. Oryzaephilus surinamensis is one of the major pests of stored spices and spice products found in many districts and countries, causing huge losses to stored products especially economical crops and food products. Oryzaephilus surinamensis management in stored food commodities is facing a challenge resulting from pesticide residues in foodstuffs and the increasing resistance of insects to conventional chemical pesticides. It is essential to develop alternative pest control techniques for protecting stored commodities. Therefore, the toxicities of different CO2-enriched atmospheres to O. surinamensis were studied, and in order to explore the action mechanism of CO2 controlled atmosphere at physiological and biochemical levels, the energy substance content and its utilization rate in O.surinamensis were also determined and calculated after stress of several hours under different CO2 concentrations or until dead. The experiments were conducted at the constant temperature of（25±1） ℃ and relative humidity of（70±5）%. Oryzaephilus surinamensis were exposed under different CO2 concentrations (10%, 30%, 50%, 70% and 90%), and the mortality of every three hours were observed until all of the insects died. In addition, the contents of polysaccharide, soluble protein and lipid in O. surinamensis were determined after the CO2 stress for 2, 4, 6, 8 h or until dead under the different CO2 concentrations, and the energy substance contents of O. surinamensis in normal atmosphere were determined to calculate the utilization rate of each energy substance. The results showed that different concentrations of CO2 had different toxicity on O. surinamensis, and the toxicity became stronger with higher CO2 concentration. The lethal time of 50% (LT50) and 99% (LT99) O. surinamensis were 52.23 and 131.45 h under 10% CO2, while they were 12.52 and 32.44 h under 90% CO2, respectively. The contents of polysaccharide, soluble protein and lipid decreased, but the utilization rate increased, with increasing CO2 concentration under the same stress time or with the extension of stress time under the same CO2 concentration, and the consumptions of the three kinds of energy substances were soluble protein ≈ lipid > polysaccharide in all the stress treatments. The residual contents of energy substance in O. surinamensis performed a gradual increase with the increasing CO2 concentration after the CO2 stress until dead, but decreased in its utilization rate. And the O. surinamensis had less residual content of energy substances under the CO2 long stress until dead than that under the CO2 short stress for 2, 4, 6 and 8 h, but it had higher utilization rate with the consumption of polysaccharide > soluble protein > lipid. Comparison of the utilization rates showed that soluble protein and lipid were the main energy substances, followed by polysaccharide, to deal with the short CO2 stress time; but polysaccharide was the prior energy substance of O. surinamensis to respond to the long stress period until dead, followed by soluble protein, and lipid was the last. So the different toxicity of different CO2 concentrations to O. surinamensis may have some relationship between different responses of insects to the energy substances and the CO2 stress, revealing that the significantly different utilization rates of energy substances in insect probably have some associations with its resistance development to CO2.

Diabetes was generally recognized as a critical issue in global public health, and has become the third noninfectious disease after angiocardiopathy and cancer. So far, the combination of applying insulin and controlling diet, as well as changing the way of living were the major therapeutic method for treating diabetes. Recently, the adjuvant therapy of natural extracts has been the research highlight in this field. To observe the intervention effect of Haematococcus pluvialis extract on blood glucose tolerance, insulin tolerance, lipid adjustment and the possible functional mechanism of improving diabetic patients’ condition, the diabetic mouse models were induced by alloxan through intraperitoneal injection, and given by gavage with different doses of H. pluvialis extract for 28 days. The contents of fasting blood glucose and their body masses were detected at 7-day intervals. The capability of blood glucose tolerance and insulin tolerance was measured during later period of administration. Meanwhile, the contents of triacylglycerol (TG), serum total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were detected. After modeling, the mice had dark and messy hair, and the typical diabetic symptoms including polydipsia, polyphagia and polyuria along with significant mass loss. Compared with the model group, by giving different doses of H. pluvialis extract in 28 d for experimental groups, the high, middle and low dose groups had statistically significant differences between the body masses and blood glucose contents, respectively (P<0.01, P<0.05, P<0.05). The difference between high dose group and model group was most evident. During the experiment of blood glucose tolerance, the blood glucose level of each group reached the peak in 30 min of glucose loading. The glucose levels appeared downtrend after 30 min, of which the high dose group and middle dose group were much lower than the model group in 120 min (P<0.01). In the insulin experiment, after injecting 0.8 U/kg insulin for 15 min, the blood glucose level of high dose group reduced by 20.9% compared with the basic level, while the low dose group only reduced by 0.6%. Three dose groups had the lowest blood glucose levels which reduced by 53.6%, 23.9% and 17.9% respectively in 90 min, while the model group reduced by 5.3%. The blood glucose increased in 120 min; however, the level in the high dose group of H. pluvialis extract was still much lower than that of model group (P<0.01). The TC, TG and LDL-C contents of the high dose group of H. pluvialis extract were much lower than the model group, which were statistically significant differences (P<0.05, P<0.01, P<0.05). Haematococcus pluvialis extract has remarkable effect of decreasing blood glucose level, relieving the symptoms of polydipsia, polyphagia, polyuria and mass loss, improving the glucose tolerance and insulin tolerance, increasing the utilization of glucose and regulating blood lipid level on diabetic mice. However, it has no effect on the blood glucose and lipids of normal mice. Among the three dose groups, the high dose group (8 g/kg) has the best effect.

In recent years, southern rice black-streaked dwarf virus (SRBSDV) has spread rapidly in Southern China and caused a serious viral dwarf disease. The white-backed planthopper (WBPH) Sogatella furcifera is a main vector for SRBSDD transmission. Use of disease resistant cultivars and insect vector control are the most economical and environmentally friendly strategy for arboviral disease control. This research focused on the resistance to SRBSDD among different rice varieties. The relationship between density of insect vectors and occurrence degree of SRBSDD was examined, and the effectiveness of disease management with controlling WBPH was measured, to build a predictive model for the occurrence of disease and damage degree of SRBSDD. In this study, we investigated the population dynamics of WBPH on 17 rice varieties in Hangzhou, China and created a predictive model for SRBSDD. The results showed that the population densities of WBPH and the disease indexes of SRBSDD on different rice varieties were significantly different, which were highest on the four rice varieties, including Yueyou 9113, Tianyouhuazhan, Y-Liangyou 2 and New-Liangyou 6. Statistical analysis demonstrated that the disease index of SRBSDD was significantly positively correlated with the population density of WBPH. For example, the disease index of SRBSDD at heading stage (Y1) was significantly positively correlated with the population density of WBPH in early August (X2) or early September (X4). The predictive model for SRBSDD was Y1=-7.962+0.110X2+2.505X4 (r=0.808, P＜0.000 6). The results confirmed the critical role of WBPH in primary infection during seedling stage to early growth stage and in secondary infection of SRBSDV, which affected the occurrence degree of SRBSDD significantly. Meanwhile, we compared the population density of WBPH and control effectiveness of SRBSDD after different pesticide treatments. The seed treatment of imidacloprid FS and the foliar application of Virtako WG 40% (chlorantraniliprole 20%+thiamethoxam 20%) decreased spread of SRBSDV by 61.7% and 73.2% respectively. The control effectiveness and disease indexes of SRBSDD showed a significant positive relationship with the population density and control effectiveness of WBPH. In conclusion, it is possible to predict the occurrence degree of SRBSDD, based on the population density of WBPH at early growth phase of rice. Using seed treatments and foliar applications of pesticides at seedling stage or early growth phase of rice is an effective way to control the disease by decreasing the major primary infection source of SRBSDV.

Tillering and earing is a key factor for yield formation in rice and one of remarkable features of high-yield colony, characterized by effective panicles per unit area. Transplanting seedling age is an important factor that may greatly affect tillering and panicle formation. Related researches have showed that by extending the transplanting seedling ages, the seedling quality would become worse; the tillering growth rate would slow down; the percentage of effective panicles would reduce; the numbers of effective panicle per plant and grains per panicle would decrease, resulting in obvious reduction of grain yield. Due to the special landscape of the hilly areas in China, the long transplanting seedling age still existed in those areas. To investigate the effect of different seedlings per hole and water-nitrogen managements on tillering and panicle formation pattern under long seedling age, F you 498 was used as test material in this experiment. A three-factor split plot was conducted with transplanting seedling ages as the main plot; the water-nitrogen management patterns as the secondary plot and seedlings per hole as the third plot. The aim was to provide the theoretical and practical data for improving the grain yield of long transplanting seedling age in hilly areas. The results showed that the tillering capacity of the older-age seedlings was stronger, and the number of leaf positions of tillers and panicles were increased. And the tillering ability of different water-nitrogen management patterns was controlled alternate irrigation pattern > dry cultivation pattern > submerged irrigation pattern under 65 d seedling age. Meanwhile, single seedling in a hole had higher percentage of emerging and earbearing tiller than double seedlings in a hole at older seedling age. At 65 d seedling age, the contribution for yield was the primary tiller > secondary tiller > main stem > third tiller; and the grain number per panicle and thousand-grain mass were primary tiller > secondary tiller > main stem, while the seed setting rate was primary tiller >main stem > secondary tiller. The superior leaf positions of emerging tiller of older seedling age were the first to ninth leaf for primary tiller, the first to second and seventh to eighth leaf for secondary tiller; the superior leaf positions of earbearing were the second, seventh to ninth leaf for primary tiller, and the first to third and seventh to eighth leaf for secondary tiller; the superior leaf positions of yield contribution were the sixth to ninth leaf for primary tiller, the first, second and seventh leaf for secondary tiller. It is concluded that the superior leaf positions of older seedling age rice are the first to second and seventh to ninth leaf, of which the total contribution to the yield reaches 68.95%. With the extension of seedling age, the yield obviously decreased; however, applying to single seedling per hole in combination with controlled alternate irrigation pattern can improve the yield of long-age seedlings effectively.

In order to reduce artificial inputs and improve the efficiency of crop production, the slow- and controlled-release nitrogen (N) fertilizer has become a research focus. However, relatively little work has been done on slow- and controlled-release N fertilizer in machine-transplanted rice. Few studies were reported on whether slow- and controlled-release N fertilizer can improve machine-transplanted rice yield and simplify the fertilization technique. Therefore, different slow- and controlled-release N fertilizers and varieties under machine-transplanted rice were set in this paper, to explore effects of slow- and controlled-release N fertilizer on N utilization characteristics and yield in machine-transplanted rice. Two-factor split-plot design was used to explore the slow- and controlled-release N fertilizer to improve N utilization efficiency and yield increasing mechanism in machine-transplanted rice. Early-maturing indica rice Zhongjiazao 17 and medium-late indica hybrid rice Chuanguyou 7329 were used as trial varieties. The main plot treatments were variety types. The subplot treatments were the coated slow- and controlled-release N fertilizer with nitrification inhibitor applied before transplanting (N1), sulfur-coated slow- and controlled-release N fertilizer applied before transplanting (N2), two resin-coated slow- and controlled-release N fertilizer applied before transplanting (N3, N4), single basal application of urea (N5), and the urea application proportion of base, tillering and spike fertilizers was 5∶3∶2 (N6). The results showed significant interactions between variety types and slow- and controlled-release N fertilizers application on biomass, N accumulation, translocation and grain yield at main growth stages. The N apparent use-efficiency was much improved when resin-coated slow- and controlled-release N fertilizer was applied. Besides, N accumulation of panicle, partial factor productivity of applied N, N agronomic efficiency, N apparent use efficiency and yield were increased by 51.83%, 18.71%, 57.97% and 5.54% compared to N6, respectively. Resin-coated slow- and controlled-release N fertilizer was the best among the slow- and controlled-release N fertilizers tested in machine-transplanted rice, which had a significant effect on N accumulation and use efficiency in medium-late indica hybrid rice. However, sulfur-coated slow- and controlled-release N fertilizer and coated slow- and controlled-release N fertilizer with nitrification inhibitor were lower than conventional fertilization in increasing yield and synergism, but they were effective for translocation of vegetative organs and N transportation efficiency. In addition, long growth period of late maturity hybrid rice Chuanguyou 7329 formed sufficient panicles and structure optimization of high quality group, and the rice could fully carry out construction of source-sink and accumulation of dry matter at full heading stage. And from full heading stage to mature stage, dry matter accumulation was improved steadily, and nitrogen accumulation increased N translocation of vegetative organs during grain filling. Ultimately, the late hybrid rice was significantly higher than early-maturing rice in N accumulation, translocation and yield at different growth stages. However, the efficiency of N utilization in Chuanguyou 7329 was low because of its low efficiency of the stem and leaf translocation. In conclusion, under the experimental condition, resin-coated N fertilizer in combination with medium-late indica hybrid rice is the optimal treatment for high yield and high N use efficiency.

In Sichuan Basin with low solar radiation, the grain yield of hybrid rice was restricted by the high humidity and small diurnal temperature. The breeding and application of large panicle hybrid varieties solved the problem to a certain extent. However, the high-yielding performance of large panicle hybrid rice was not stable. In order to explore the measures of maintaining the high-yielding performance of large panicle hybrid rice, it is necessary to investigate the characters of its grain yield formation. Two hybrid varieties with different panicle masses (Chuannongyou 498, about 5 g per panicle; Chuanyou 6203, about 4 g per panicle) were studied on the differences of dry matter accumulation and yield components, and five different nitrogen (N) managements (the N application proportion of base-tillering and panicle fertilizers was 90∶10, 75∶25, 60∶40, 45∶55, 30∶70, respectively) integrated with 15N tracing were applied to investigate the traits of N uptake across the whole growth stage. The results showed that there was no significant difference in spikelet per panicle between large and medium panicle hybrid rice. Medium panicle hybrid rice showed significant advantages in effective panicles and total spikelet compared to large panicle hybrid rice; however, the thousand-grain mass of large panicle hybrid rice was larger, and this superiority played a major role in gaining higher grain yield than medium panicle hybrid rice. There was no significant difference in dry matter accumulation at maturity stage between large and medium panicle hybrid rice, but difference of dry matter productivity was observed before and after anthesis. Compared to medium panicle hybrid rice, large panicle hybrid rice accumulated more dry matters before anthesis, and larger percentage of which was exported to the panicle at anthesis, contributing approximately 40% of its grain yield. For medium panicle hybrid rice, dry matter exportation only contributed about 25% of its grain yield. For N management treatments, 40% and 25% were the optimal panicle N ratios which could gain the highest grain yields for large and medium panicle hybrid rice respectively. According to the relation formula between panicle N ratio and grain yield, 33% was the optimal panicle N ratio which produced the most grains for large panicle hybrid rice, while the optimal panicle N ratio for medium panicle hybrid rice was 27%. The difference for the optimal panicle N ratio between large and medium panicle hybrid rice was caused by varied demands on N at different growth stages. There was no significant difference for N uptake of basal fertilizer between large and medium panicle hybrid rice. Medium panicle hybrid rice showed significant merits in assimilating N at tillering stage than large panicle hybrid rice. In contrast, large panicle hybrid rice assimilated more N at booting stage. A further increase of panicle N based on the optimal panicle N ratio could increase N recovery efficiency, but N physiological efficiency would drop rapidly and the yield-increasing effects of N fertilizer became poor. In conclusion, 33% and 27% are the optimal panicle N ratios for large and medium panicle hybrid rice, respectively, which increase the grain yield and N utilization efficiency simultaneously.

In order to reveal spatial and temporal variation and related influencing factors of rice yield in Southwest China, the rice production data were analyzed in four administrative regions including Yunnan, Guizhou, Sichuan and Chongqing during 1978—2013 years. The results showed that the rice production increased by 40.0% during 1978—2013 years in Southwest China, with the higher increase during 1980s—1990s and keeping relatively stable after 1990. Meanwhile, rice planting area was reduced by 11.0%, which indicated an adverse condition that may restrict sustainable rice production. Rice planting area in Sichuan accounted for more than 45% in Southwest China and rice planting area in Yunnan showed increasing trend since 2000, which was the basic condition that ensured large increase in rice production during 1980s—1990s. Rice yield improvement was a key factor to promote higher rice production; however, the rice yield was still lower than 7 000 kg/hm2. Climate change posed an adverse effect on higher rice yield in this stage, particularly in Sichuan and Yunnan. Taken together, basic measures should be carried out to ensure the sustainable rice production such as inhibiting planting area reduction in Sichuan, Guizhou and Chongqing, keeping steady increase in Yunnan. In addition, major ways should be taken to breed high-yielding varieties with stress resistance, to create new cultivation techniques in combination with prevention and reduction of natural disasters in rice production, particularly in Sichuan and Yunnan.

Planting Caragana has achieved remarkable success in the recovery of desertification land in desert steppe because of its excellent biological characteristics, such as tolerance of sand burying, drought-resistance, and so on. Thus, its sustainability of ecological restoration becomes one of the key topics of concern. Restoration of soil habitat is the foundation for ecosystem being fully restored, and soil texture is the core indicator to evaluate soil properties. In this study, soil texture evolution process and effects after planting C. intermedia stand were investigated to understand its role in desert steppe ecosystem. Series of different stand ages (10 a, 17 a, 27 a) and space sequences (from the inside via the edge to the outside of shrubs) were designed to analyze the spatiotemporal rules of soil particle composition, increment in silt and clay, volume fractal dimension of soil particles, as well as the contents of soil organic carbon (SOC) and soil total nitrogen (STN)． The results showed that: 1) The percentages of fine sand (ranging from 0.05 mm to 0.1 mm) were higher than those of silt and clay, coarse sand and gravel sand in the soil particle composition. With the increase of C. intermedia stand ages, soil texture was improved gradually according with vertical orientation of soil depth from top to bottom and horizontal direction from the inside to the outside of shrubs. The volume fractal dimension of soil particles inside shrubs increased by 0.189±0.401 on average compared to the outside. 2) Spatial heterogeneity of SOC and STN in the surface soil layer changed significantly with the increase of C. intermedia stand ages. SOC and STN inside shrubs increased by (0.087±0.100)% and (0.008±0.010)% respectively in comparison to those outside shrubs, which indicated the fertility island effect by C. intermedia. The contents of SOC and STN were significantly and positively correlated with the content of silt and clay (P<0.01). 3) Caragana intermedia plantation exerted a distinct co-succession influence on the content of silt and clay and the resource of carbon and nitrogen in desert steppe. In conclusion, soil texture and soil fertility states are significantly improved after the construction of Caragana plantation. However, soil texture is improved more slowly when plantation aged.

The technology for microorganism immobilization originated from the immobilized enzyme technology in the 1970s. After decades of development, it has been widely used in food fermentation and environmental protection, etc. Especially, microbe-embedded capsules have drawn substantial attentions from researchers due to their significant roles in water purification and sewage treatment. However, lack of consolidated standards has impeded their applications. Texture profile analysis (TPA) has been widely used in food industry for determination of product structure and quality, while its application in non-food field is yet to be explored. Therefore, in the present study, TPA was used for the development and optimization of bacteria-embedded solid capsule systems in the purpose of water purification. To achieve this purpose, solid capsules were prepared with different materials, such as diatomite, zeolite powder and bamboo charcoal in different blending ratios. TPA was employed to characterize these capsules in respects of hardness, cohesiveness and elastic resilience (springiness). Meanwhile, scanning electron microscopy (SEM) was adopted to investigate the sectional structure and pore size of these capsules. Furthermore, simulative experiments in both ultrapure water and sewage were carried out to examine the releasing velocity and water purification efficiency of these bacteria-embedded solid capsules. Combining with TPA and SEM, the factors influencing the internal structure of capsules including embedding/crosslinking medium, curing duration, and ingredient proportions were explored, thus providing standards and guidelines for the preparation of bacteria-embedded capsules for large-scale practical applications in the future. The TPA data showed that a high ratio of sodium alginate in the embedding medium resulted in capsules with high strength, and polyvinyl alcohol rendered cohesiveness and resilience (springiness) to capsules. An optimal embedding medium system was established as follows: V(4% sodium alginate) ∶V(10% polyvinyl alcohol)=1∶9 for embedding medium, while the mixture of 4% boric acid and 4% calcium chloride for crosslinking medium. The optimal concentrations of various materials were determined as follows: 20%30% for diatomite, 20% 80% for zeolite powder and 20%60% for bamboo charcoal. The optimal curing duration varied from 16 to 36 h. The SEM images indicated the existence of internal microspores both at nanoscale and micron-size in the crosslinked capsules, and differences on microstructures of capsules were observed among single or combining addition of diatomite, zeolite powder and bamboo charcoal. Both specific surface area and pore volume decreased after embedding, but not for pore size. The releasing rates of bacteria in capsules in 24 h were low, from 1% to 14%. In the simulative experiments of the sewage purification, all groups, including bacteria-embedded capsule and free bacteria, showed good ammonia removal efficiency. The final concentration of ammonia was below 0.1 mg/L after 20 h, while the initial concentration was 39.3 44.7 mg/L. The bacteria-free group had the highest ammonia removal efficiency, followed by mixture 1 group (1 mL of 4% sodium alginate, 9 mL of 10% polyvinyl alcohol, 1.5 g diatomite, 0.5 g zeolite powder, 0.5 g bamboo charcoal, and 2 mL bacterial culture) and bamboo charcoal group. Meanwhile, the total nitrogen concentration decreased at a certain degree. These results confirmed that these capsules were applicable for sewage treatment. In conclusion, the present study establishes successfully standards and guidelines for the preparation of bacteria-embedded capsules on the basis of TPA in combination with SEM and simulative experiments. This profound step can accelerate the practical application of bacteria-embedded capsules in actual sewage purification.

Caragana intermedia is a leguminous shrub plant, which is planted in desert steppe area on a large scale, which can also be used as forage grass, with very important ecological and economic value. However, a series of shrub and grass resource competition problem emerged with the aging of C. intermedia shrublands. The growth of zonal perennial vegetation often subjected to great constraints, and self-degradation of C. intermedia shrublands was also developed to different degrees. The reasonable collocation relations of shrub and grass were one of the key elements to maintain ecology and production services of C. intermedia shrublands in desert steppe. The objective of this study is to understand composition characteristics of herbaceous plant species and its dynamic change rules around the C. intermedia shrublands, and to reveal the structure of shrub and grass and the evolution trend of C. intermedia shrubland system. Taking natural grassland as a control, spatiotemporal substitution methods were used, and a research sequence constituted of different ages of C. intermedia shrubland plots was erected to carry out tracking, monitoring and analysis of vegetation seasonal dynamics at interior and exterior of the shrubs. The results showed that: 1) The number of natural grassland (CK) and herbaceous plant species of C. intermedia shrublands were 25 species, belonging to 8 families and 23 genera. The highest species number was the 16 a C. intermedia shrublands (16 species), and the number of species gradually decreased with aging of C. intermedia shrublands. Pennisetum centrasiaticum, Cleistogenes squarrosa and other perennial grasses began to appear, but the dominant species still was short-lived annual plants. Species diversity indexes showed a decreasing trend after a steady increase with aging of C. intermedia shrublands. 2) The seasonal dynamics, coverage degree, aboveground biomass and species number of herbaceous layer vegetation were mainly affected by the change of precipitation and temperature, but the existence of C. intermedia shrubs delayed the growth recovery rate of herbaceous layer vegetation after spring drought, and the highest relative volatility index was 25 a C. intermedia shrublands. 3) The coverage degree of herbaceous layer vegetation at the interior and exterior of the C. intermedia shrubs and the seasonal dynamics of aboveground biomass at the interior of the C. intermedia shrubs were significantly changed by the ages of C. intermedia shrubland, and a significant interaction between ages of C. intermedia shrublands and seasonal factors was observed.

Silky Fowl is a precious local breed containing a large amount of melanin. Therefore, it is considered to have high edible and medicinal value. Study on the related gene expression is helpful to reveal the internal regulation mechanism of melanin deposition. TYR (tyrosinase), MITF (microphthalmia-associated transcription factor) and ASIP (agouti signaling protein) are important candidate genes for the formation of pigment. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) has been widely applied to analyze gene expression as a convenient approach, and the selection of appropriate reference genes according to specific samples or conditions is critical to ensure the accuracy and reliability of gene expression. The objectives of this study are to screen the ideal reference genes and to provide a necessary basis for further study on gene expression regulation of melanin deposition and other important traits in Silky Fowl through investigating the expression stability of reference genes (ACTB, GAPDH and 18S rRNA) and their effects on the expression of melanin genes (TYR, MITF and ASIP) in tissues of Silky Fowl. RT-qPCR was adopted to investigate mRNA expression levels of the related genes in the muscle, kidney, liver, gizzard and skin of Silky Fowl, and the ultraviolet spectrophotometry was used to measure the melanin content in tissues of Silky Fowl. GeNorm and NormFinder softwares were applied to evaluate the expression stability of reference genes, and 2-△△CT method was applied to calculate the relative expression of target gene. The results from RT-qPCR showed that the melting curve displayed an obvious single peak and standard curves had good linear relationship. Comprehensive evaluation of geNorm and NormFinder indicated that the expression stability of GAPDH was the highest, while ACTB was the lowest among the three reference genes in tissues. Relative quantity and melanin content analysis demonstrated that taking GAPDH as a reference gene, the expression level of TYR, ASIP and MITF genes with the melanin content in tissues had the same trend: skin > kidney > gizzard > liver > muscle, but the expression level of ASIP gene was opposite to melanin deposition content, which implied that each target gene expression was matched with their respective function on regulating melanin deposition. For instance, high expression of TYR and MITF genes promoted melanin deposition, while the ASIP gene inhibited melanin deposition. Taking 18S rRNA and ACTB as reference genes, the expression of TYR, MITF and ASIP genes were not related to the melanin deposition, and an ideal calibration result could not be obtained. In conclusion, reference gene plays an important role in the expression of target gene. In this paper, GAPDH gene not only had highest expression stability in tissues, but also could truly reflect the regulation effect of the target genes (TYR, MITF and ASIP) on melanin deposition. Thus, GAPDH gene is the most suitable reference gene for normalizing the mRNA expression of genes in tissues of Silky Fowl.