通过PCR扩增出洋葱伯克霍尔德氏菌Lu10-1脂肪酶基因,将基因片段分别克隆到大肠杆菌、毕赤酵母和枯草杆菌的表达载体,转入表达菌株中.结果表明,重组脂肪酶在大肠杆菌中主要以包涵体形式表达,在毕赤酵母中未有表达,而在枯草杆菌中实现了胞外分泌表达,测得发酵上清液酶活为13.8 U/mL.对重组枯草杆菌发酵条件进行了摇瓶初步优化和3L的反应器分批培养.当以TB为出发培养基,初始pH 6.5,温度为37℃时,在3L的发酵罐上最终酶活达到34.5 U/mL,是野生菌表达量的4.2倍.

It is aimed to optimize the fermentation conditions of lipase-producing Burkholder cepacia Lu10-1 and analyze its enzymatic properties. The single factor and orthogonal experiments were adopted to study the major factors that affected the lipase yield, such as carbon, nitrogen, inducer, initial pH, temperature, etc. The impact of temperature, pH, metal ion, and organic solvents on lipase catalytic reaction was also primarily investigated. The optimal medium and culture conditions were as follows:soluble starch 1.5%, peptone 1.5%, olive oil 3 g/L, K2HPO4 2 g/L, temperature 32℃, initial pH 9.0, and fermentation time 48 h, the enzyme activity reached 12.4 U/mL, increased 2.7-fold compared to the initial activity. The optimal temperature and pH were 60℃and 9.0 respectively. The lipase was stable under 60℃for 100 h remaining over 80%of the activity and it was also stable at pH 5.0-10.0. Moreover, the lipase had solid tolerance to organic solvent such as methanol and ethanol.

Diabetes mellitus is a clinically complex disease characterized by chronic hyperglycemia with metabolic disturbances. In this study, we investigated the effect of mulberry leaf polysaccharide (MLPII) on pancreatic islet cell apoptosis and insulin secretory function in diabetic rats induced by a high fat diet and streptozotocin. Our results showed that MLPII treatment inhibited pancreatic islet cell apoptosis and ameliorated insulin secretory capacity of pancreatic β-cells in diabetic rats. And further study demonstrated that chronic treatment of diabetic rats with MLPII resulted in up-regulation of anti-apoptotic B-cell leukaemia/lymphoma 2 (Bcl-2) protein and down-regulation of pro-apoptotic Bcl2-associated X (Bax) and caspase-3 protein in pancreatic islet cells. Moreover, MLPII significantly restored pancreatic duodenal homeobox-1 (PDX-1) protein nuclear localization, and increased mRNA and protein expression of PDX-1 and its downstream targets, glucose transporter 2 (GLUT2) and glucokinase (GCK) in pancreatic islet cells of diabetic rats. These findings suggested that MLPII might play a critical role in protecting pancreatic islet cell from apoptosis via elevation of Bcl-2/Bax ratio, and ameliorating insulin secretory capacity of pancreatic β-cells via restoration of PDX-1 nuclear localization and expression levels in diabetic rats. This is the first report to explore the potential molecular mechanism involved in the hypoglycemic activity of the polysaccharide from mulberry leaves.

Mulberry leaf is a traditional medicine used to treat diabetes in the clinic. The aim of this study was to determine the mechanisms by which mulberry leaf polysaccharide (MLPII), improves hepatic glucose metabolism and insulin resistance in rats with type 2 diabetes induced by high fat and streptozotocin (STZ). MLPII was administered for 6 weeks after establishment of type 2 diabetes in Wistar rats. At the end of the experiment, oral glucose tolerance, liver glycogen content, glucose synthase (GS) activity and insulin resistance were determined. Expression patterns of proteins and genes associated with insulin signaling as well as biomarkers of oxidative stress and antioxidant enzyme activities were assayed. Compared with normal control rats, MLPII treatment significantly improved oral glucose tolerance (P<0.01) and restored the glycogen level (P<0.01) and GS activity (P<0.05) in diabetic rats. Insulin resistance was improved in MLPII-treated diabetic rats (P<0.01). Furthermore, expression levels of insulin receptor substrate 2 (IRS2), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB/AKT) involved in insulin signaling were significantly increased (P<0.01), while protein–tyrosine phosphatase 1B (PTP1B) expression was markedly reduced (P<0.01). The levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) in livers of the MLPII-treated group were significantly reduced (P<0.01), while activities of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), were significantly increased (P<0.01, P<0.01, P<0.01, respectively). The results clearly indicate that MLPII treatment effectively normalizes hepatic glucose metabolism and insulin signaling by inhibiting the expression of PTP1B, activating the PI3K–AKT pathway and mitigating oxidative stress in the livers of rats with type 2 diabetes induced by high fat and STZ.

In the present study, a high-purity polysaccharide from mulberry leaf (MLP) was purified and characterized, and its anti-diabetic effects were investigated in streptozotocin (STZ)-induced diabetic rats. Our results showed that the obtained MLP (purity 99.8%) was determined to be composed of d-arabinose, d-xylose, d-glucose, d-rhamnose and d-mannose with molar ratio of 1:2.13:6.53:1.04:8.73. Oral administration of MLP at 50–200 mg/kg body weight daily for 5 weeks significantly reduced the levels of fasting blood glucose (FBG), glycosylated serum protein (GSP), serum total cholesterol (TC), and serum triglyceride (TG), and increased the body weight, fasting insulin (FINS), C-peptide (C-P), liver glycogen, liver glucokinase, and serum high-density lipoprotein cholesterol (HDL-C). Moreover, MLP promoted marked pancreatic β-cell regeneration and insulin secretion, and reduced liver fat accumulation in diabetic rats. The treatment effect of MLP on diabetes was similar to the effect of antidiabetic drug glibenclamide. These results clearly indicated that MLP may have a potential for the treatment of hyperglycemia and hyperlipidemia in diabetes.

MLPⅡ,a uniform polysaccharide ingredient isolated and purified from mulberry(Morus L.) leaves,possesses hypoglycemic activity.In this study,we investigated the improvement effect of mulberry leaf MLPⅡ on hepatic insulin resistance in diabetic model rats through measuring and analyzing the insulin sensitive index and hepatic physiological indicators of diabetic model rats in the MLPⅡ-treated group.Compared with model rats in the diabetic control group,the content of total cholesterol(TC),triglyceride(TG) and malondialdehyde(MDA) in liver tissue of the diabetic model rats in the 150 mg/kg MLPⅡ-treated group was reduced by 40.59%,38.58% and 52.81% respectively,while superoxide dismutase(SOD) activity,glycogen content and glucokinase activity was increased by 15.88%,198.48% and 169.77% respectively,all of which reached extremely significant level.In addition,compared with model rats in the diabetic control group,the insulin sensitive index of diabetic model rats in all MLPⅡ-treated groups was obviously improved,and the degree of hepatic adipose degeneration was obviously alleviated,both reached extremely signifi-cant level in the 150 mg/kg MLPⅡ-treated group.These results suggest that mulberry leaf polysaccharide MLPⅡ can significantly improve hepatic glucose and lipid metabolism,and hepatic insulin resistance in diabetic model rats,and this effect is probably associated with the regulation of insulin release and the response level to oxidative stress in the liver.

Odorant binding proteins (OBPs) in insects play an important role in foraging,courtshipping,multiplying and chemical communication with the environments. Antennal-binding protein (ABP) is one of important groups of OBP family. We analyzed the first reported ABP and ABPX genes in the silkworm Bombyx mori using chromosome mapping and semi-quantitative RT-PCR for further understanding of the expression and function of ABP/ABPX. Chromosome mapping showed that BmABP and BmABPX were respectively organized in chromosome 5 and 26 with different gene structure,suggesting that they probably possess different functions. Analysis of the expression profiles in different tissues of males and females during the embryonic,larval and adult stages revealed that BmABP possessed high expression level in a variety of tissues and organs with no time- and tissue-specificity,while BmABPX possessed conspicuous temporal and spatial expression differences (P 0. 05) ,with the highest relative expression level in the antenna,and lower relative expression levels in most other non-olfactory tissues without significant sex differences. The results suggest that these two genes are more likely to have other functions undiscovered besides olfactory-related functions.

MLPⅡ,the purified polysaccharide ingredient isolated from mulberry(Morus L.) leaves,possesses hypoglycemic activity.High performance liquid chromatography analysis showed that mulberry leaf polysaccharide MLPⅡ was mainly composed of mannose(man),rhamnose(rha),glucose(glc),xylose(xyl) and arabinose(ara) with the molar proportion of 8.73 ∶1.04 ∶6.53 ∶2.13 ∶1.00.Infrared spectrometry analysis showed that polysaccharide MLPⅡ was mainly linked by β-glycosidic bond.The experimental results of hypoglycemic effect of polysaccharide MLPⅡ on diabetic rats indicated that,compared with the diabetic control group,body weight of diabetic rats in the 150 mg/kg MLPⅡ-treated group increased by 26.22%,while fasting blood glucose(FBG) decreased 52.89%,both of which reached extremely significant level.In addition,improvement on levels of glycosylated haemoglobin(HbA1c),fasting insulin(FINS),and C-peptide(C-P) were observed in the MLPⅡ-treated diabetic rats.The damage of pancreatic cells were repaired,and the insulin synthesis and secretion in pancreatic β-cells were enhanced following treatment with mulberry leaf polysaccharide MLPⅡ.These results suggest that mulberry leaf polysaccharide MLPⅡ could effectively control the levels of body weight and FBG levels,and ameliorate the glucose tolerance of diabetic rats.Moreover,mulberry leaf polysaccharide MLPⅡ exerts the antihypergly-cemic action at least in part by promoting insulin synthesis and secretion in diabetic rats.

Macrophomina phaseolina MOD-1 is an endophytic oleaginous fungus isolated from the healthy mulberry root.To optimize medium composition and fermentation conditions for lipid production from MOD-1,the experiments with single factor and uniform design were conducted.The best medium composition was found with soluble starch 105 g/L,peptone 1.1 g/L,KH2PO4 1.5 g/L,(NH4)2SO4 0.3 g/L,MgSO4 0.32 g/L,and MnCl2 4.9 nmol/L.Based on this culture medium,the optimal fermentation conditions were as follows: initial pH 7.0,temperature 26 ℃,rotation speed 190 r/min and medium volume 100 mL.After being cultured for 6 h under the optimized condition,the culture's dry cell weight was found as high as 41.852 g/L and lipid yield reached 25.511 g/L.Determination of fatty acid composition in the cultures obtained under optimized culture medium or fermentation condition by gas chromatography and mass spectrometry(GC-MS) revealed that there were 9 kinds of fatty acids in the lipids from the former condition,among which the content of monounsaturated fatty acids was 67.92%,and there were 7 kinds of fatty acids in the lipids from the latter condition,among which the content of monounsaturated fatty acids was 74.32%.The results indicate that,under optimized condition,lipid yield of MOD-1 is increased.Its lipid composition is simple and easy to be purified.Besides,it has a high content of monounsaturated fatty acids.

BACKGROUND:Anthracnose, caused by Colletotrichum dematium, is a serious threat to the production and quality of mulberry leaves in susceptible varieties. Control of the disease has been a major problem in mulberry cultivation. Some strains of Burkholderia cepacia were reported to be useful antagonists of plant pests and could increase the yields of several crop plants. Although B. cepacia Lu10-1 is an endophytic bacterium obtained from mulberry leaves, it has not been deployed to control C. dematium infection in mulberry nor its colonization patterns in mulberry have been studied using GFP reporter or other reporters. The present study sought to evaluate the antifungal and plant-growth-promoting properties of strain Lu10-1, to clarify its specific localization within a mulberry plant, and to better understand its potential as a biocontrol and growth-promoting agent.RESULTS:Lu10-1 inhibited conidial germination and mycelial growth of C. dematium in vitro; when applied on leaves or to the soil, Lu10-1 also inhibited the development of anthracnose in a greenhouse, but the effectiveness varied with the length of the interval between the strain treatment and inoculation with the pathogen. Strain Lu10-1 could survive in both sterile and non-sterile soils for more than 60 days. The strain produced auxins, contributed to P solubilization and nitrogenase activity, and significantly promoted the growth of mulberry seedlings. The bacteria infected mulberry seedlings through cracks formed at junctions of lateral roots with the main root and in the zone of differentiation and elongation, and the cells were able to multiply and spread, mainly to the intercellular spaces of different tissues. The growth in all the tissues was around 1-5 × 105 CFU per gram of fresh plant tissue.CONCLUSIONS:Burkholderia cepacia strain Lu10-1 is an endophyte that can multiply and spread in mulberry seedlings rapidly and efficiently. The strain is antagonistic to C. dematium and acts as an efficient plant-growth-promoting agent on mulberry seedlings and is therefore a promising candidate as a biocontrol and growth-promoting agent.

L144 strain,isolated from the healthy mulberry leaves,was an antagonistic bacterium that exhibited excellent resistant against various plant pathogens.The strain was identified as Bacillus subtilis based on the result of its 16S rRNA gene sequence homology,and the physiological and biochemical characteristics.The sequence of 16S rDNA obtained in this study had been registered at GenBank database(Accession number EU118756).The determination results of biological characteristics showed that L144 strain could use many substances as the sources of carbon and nitrogen,and the optimal pH and temperature for the growth were 6.5 and 33 ℃,respectively.

OBJECTIVE To identify and colonize an antagonistic bacterium, Lu10-1, isolated from the healthy mulberry. METHODS Strain Lu10-1 was identified based on the analysis of its 16S rRNA gene sequence homology, the physiological and biochemical characteristics, and the recA gene sequence comparison. A spontaneous Lu10-1 mutant tolerant to rifampicin and ampicillin were isolated by gradually increasing the concentration of the two antibiotics. The mutants were used to assess the ability of Lu10-1 to colonize mulberry by different inoculation approaches, including stem and leaf acupuncturing, seed soaking, root soaking and leaf daubing. RESULTS Lu10-1 belonged to Burkholderia. In the phylogenetic tree, Lu10-1 was the closest relative to B. cepacia (X80284) with more than 98% sequences similarity. The 16S rDNA sequences of Lu10-1 have been registered at GenBank database under the accession number EF546394. Moreover, our results also indicated that the population of strain Lu10-1 living in the mulberry tissues decreased as a whole after the treatment of seed soaking. The bacterial density inside the mulberry seedling tissues decreased to a steady level 20 days after germination. The population of strain Lu10-1 in mulberry leaves and stems after the treatment of root soaking increased first and then decreased. CONCLUSION The strain Lu10-1 fell into Burkholderia cepacia genomovar I as a single species. Furthermore, the strain Lu10-1 could colonize and transmit in mulberry, while its resistance to plant pathogen was not changed during the process of colonization compared to the original strains. Taken together, we suggest that Burkholderia. cepacia Lu10-1 will play an important role in the biological control of mulberry disease.

Forty-five bacterial isolates were collected from surface-sterilized leaves of mulberry (Morus alba L.). By screening their antagonistic activities against Ralstonia solanacearum in vitro, four isolates showed a remarkable inhibitory effect. The evaluation of the antagonistic strains against bacterial wilt of mulberry indicated that the strain Lu144 effectively reduced disease incidence. In the greenhouse, Lu144 displayed effective biological control against bacterial wilt of mulberry when it was applied to sterile or nonsterile soil before the infection by the pathogen. Based on bacteriological properties and 16S rRNA gene sequencing, Lu144 was identified as a strain of Bacillus subtilis. The endophytic population and infection process of Lu144 in mulberry seedlings was explored following recovery of the green fluorescent protein (GFP)-labeled Lu144 and examination of the labeled strain by confocal laser scanning microscopy. Interestingly, the infection of GFP-labeled Lu144 cells into the mulberry seedlings occurred through the cracks formed at the lateral root junctions and the zone of differentiation and elongation, and the cells were able to develop and transfer in mulberry and mainly in the intercellular spaces of different tissues. The population of the GFP-labeled Lu144 inoculant was larger and more stable in leaves than that in roots and stems.

One hundred and fourteen strains of endophytic fungi were isolated from the roots,stems and leaves of different mulberry varieties using the tissue culture method.Through the morphological identification,they belonged to 21 genera,including Fusarium,Alternaria,Scytalidium etc.The result showed endophytic fungi in mulberry exhibited the broad biodiversity.The distribution of endophyte was different among mulberry varieties.The type and quantity of endophytic fungi had great difference among different organs of the same variety:rootsstemsleaves.Fusarium was the dominant population in roots,while Alternaria was the dominant population in stems and leaves.Monilia and Fusarium were isolated from three organs,and the others were isolated from one or two organs.

This paper reviews the infection and colonization process of endophytic bacteria in detail,namely three stages(adsorption,incursion and colonization),with particular reference to the colonization detected methods,such as labelled means of antibiotic,the technique about immunology,marker gene and the characteristic oligomonophosphate segment tag etc.In addition,the origin and colonial impact factor of endophytic bacteria are systematically discussed,with expectation in the future studies on the colonization of endophytic bacteria.

229 endophytic bacteria strains were isolated from the tissues of different healthy mulberry varieties.The reasonable method for isolation of endophytic bacteria and purification was discussed, and the amount of endophytic bacteria was determined.The results showed that endophytic bacteria exist in all mulberry tissues including root,stem,leaf stalk,lamina and flower bud.But the population densities vary with varieties,tissues and growth periods of the plant.In vitro antagonistic examination revealed that in all 229 tested strains 42 strains(18.3%) showed antibiosis activities against Colletotrichum morifolium Hara,25 strains(10.9%) showed antibiosis activities against Aecidium mori(Bard) Diet and 8 strains showed strong inhibitory activities against some plant fungal pathogens,revealing potential in the aspect of antagonistic action.Bacteriological analysis showed that these 8 strains were belong to Bacillus sp.(G21,G49,Y12 and J26),Pseudomonas sp.(G82 and J50),Erwinia sp.(Y33)and Curtobacterium sp.(B19),respectively.

以9株白僵菌菌株为材料,对其进行鉴定并探讨了菌株的生物学特性。结果表明,有2株为卵孢白僵菌,7株为球孢白僵菌;以营养生长量、产孢量、抗紫外线能力和致病力等生物学指标进行综合比较筛选,球孢白僵菌B1为生产白僵蚕的最佳接种菌株。

To understand the role of bacteria from the silkworm intestine in the digestion and assimilation of food,and to screen potential beneficial strains for the future applications in the silkworm rearing,30 strains were isolated from the intestine of silkworm and their enzyme-producing capabilities were studied.Results showed that a strains were able to produce protease,7 strains were able to produce either lipase and 7 strains produced cellulose,and 8 strains were able to produce amylase(8 stains).Among the 30 isolates,1 strain excreted all four enzymes,4 strains excreted three kinds of enzymes,2 strains excreted two kinds of enzymes,11 strains excreted one enzyme,and other 12 strains didn't excret any enzyme.In other words,60% of the isolates were capable of producing enzymes.Therefore,it can be concluded that bacteria in the intestine of silkworm play an important role in the digestion of food.

Using orthogonal experiment design,the effect of Thidiazuron(TDZ) and α-Naphthalencacetic acid(NAA) content in the medium, effect of the order of leaf,the age of leaf and the varieties of mulberry on the leaf callus induction was investigated.Meanwhile,the effect of 6-benzylaminopurine(BA),indole-3-acetic acid(IAA) and sugar on callus differentiation was also studied.The results showed that the best condition for callus induction is: age of the mulberry cultured in vitro,21 d;medium,MS(Murashige & Skoog medium)+1.0 mg/L TDZ+0.2 mg/L NAA;and that the medium for adventitious bud differentiation of the callus is: MS+3.0 mg/L 6-BA+0.1 mg/L IAA,fructose,2%.Under these conditions,the leaf callus induction rate and adventitious bud differentiation rate for Shan 305 reached 93.9% and 100% respectively.