Cloning and expression analysis of ScBAK1 gene and its alternative spliceosome in sugarcane北大核心CSCD

Alternative splicing (AS) is an important part of regulation of eukaryotic gene expression. BAK1 (Brassinosteroid insensitive1-associated receptor kinase 1) is a specific type of plant serine/threonine protein kinases, and can regulate growth and development and natural immunization. To reveal the responses of sugarcane BAK1 gene to the adverse environment, a ScBAK1 gene and its alternative spliceosome, termed ScBAK1 (GenBank accession number: KP032226) and ScBAK1 S1 (GenBank accession number: KP032227), were cloned from leaf cDNA of Yacheng 05-179 utilizing the methods of electronic cloning and RT-PCR. The open reading frame (ORF) length of ScBAK1/ScBAK1 S1 gene was 1 860bp/1 770bp, encoding 619/589 amino acids residues. The predicted molecular weight of the protein was 69.28 kDa/ 65.76 kDa. Both proteins were located in plasma membrane, estimated as acid, hydrophikic and secretive proteins. Random coil and alpha helix gave priority to extended strand in their secondary structure without beta turn. The most important protein function was cell envelope, secondly biosynthesis of amino acids and cofactors. Real-time quantitative PCR analysis revealed that the expression of sugarcane ScBAK1 S1 gene exhibited the reduced expression trend under smut fungus stress and various abiotic exogenous stresses, including SA, CuCl2, PEG, ABA, NaCl and JA, while the expression of ScBAK1 gene was induced by SA, CuCl2, PEG, NaCl and smut fungus stresses. The phenomenon showed that the absent sequences or amounts of ScBAK1 S1 gene plays a key role in the response of ScBAK1 to the stress of sugarcane smut fungus, osmotic stress and cell growth. The differential expression of ScBAK1 and ScBAK1 S1 lays a foundation for further research on the function of ScBAK1 gene under biotic and abiotic stress.     

Cloning,expression, and functional identification of CaSWEET7 and CaSWEET15 genes in Canarium album北大核心CSCD

糖外排转运蛋白(sugars will eventually be exported transporters,SWEET)介导植物光合同化产物蔗糖的跨膜运输.以橄榄(Canarium album(Lour.)Raeusch.)果实为材料,通过气相色谱串联质谱(GC-MS)检测橄榄果实中糖组分及含量变化,利用RT-PCR技术克隆得到CaSWEET7和CaSWEET15基因开放阅读框(ORF)序列.结果表明:CaSWEET7和CaSWEET15基因ORF全长分别为774 bp和951 bp,分别编码长度为257个和316个氨基酸残基,具有2个MtN3_slv结构域;进化树分析表明,CaSWEET7属于CladeⅡ,CaSWEET15属于CladeⅢ.实时荧光定量PCR(qRT-PCR)技术检测其在不同发育时期的果实中相对表达量变化,结果表明,随着果实发育,CaSWEET7和CaSWEET15表达量逐渐递增,且与果实发育蔗糖含量变化呈显著正相关(相关性系数分别为0.931和0.904,P<0.05);利用酵母功能互补证明CaSWEET7和CaSWEET15基因编码的蛋白具有转运蔗糖能力.本研究表明CaSWEET7和CaSWEET15基因可能在橄榄果实蔗糖积累中发挥作用.(图9表2参44)     

Cloning and expression of RING-finger E3 ubiquitin ligase CsSDIR1 gene in tea plant (Camellia sinensis)北大核心CSCD

Protein ubiquitination regulates many aspects of plant development and stress response. The RING-finger type E3 ubiquitin ligase SDIR1 (salt and drought induced ring finger 1) gene plays a key role in plant stress response. In this study, the full-length cDNA and the promoter sequences of CsSDIR1 were isolated from tea plants using the RT-PCR technology, and its bioinformatics characteristics were systematically analyzed. The expression patterns of CsSDIR1 in various tissues and in response to cold, drought, salt, and ABA treatments were also investigated by quantitative real-time RT-PCR (qRT-PCR). The sequence of CsSDIR1 contains a complete open reading frame of 831 bp, coding for a 276-long amino acid protein with a molecular weight of (Mr) 30.085 × 103 and a theoretical isoelectric point of 6.54. CsSDIR1 was predicted to be a hydrophobic protein localized on the intracellular membranes. The analysis of the amino acid sequence characteristics showed that CsSDIR1 contains two putative transmembrane domains at the N-terminus and a C3H2C3 RING-finger domain at the C-terminus; it shares high similarity with other plants' SDIR1, and has the closest relationship to Actinidia sinensis. A cis-acting regulatory elements prediction showed that the CsSDIR1 promoter contains many cis-acting elements, especially drought and salt stress response elements. The qRT-PCR analysis indicated that the CsSDIR1 gene has a high expression level in stems, followed by roots, leaves, and flowers; the expression of the CsSDIR1 gene is up-regulated by ABA, salt, and drought treatments, whereas it is down-regulated in response to cold stress. These results demonstrated that the CsSDIR1 gene might be involved in the plant stress response of tea trees. © 2018 Science Press. All rights reserved.     

Cloning and expression analysis of geosmin synthase gene from Aphanizomenon gracile北大核心CSCD

Geosmin is a secondary metabolite responsible for earthy odors. The occurrence of geosmin has great impact on the quality of water environment. The gene essential for geosmin biosynthesis have been identified in several species. But little is known about the mechanism of geosmin synthesis in Aphanizomenon gracile. This study attempted to clone the gene involved in geosmin biosynthesis of Aphanizomenon gracile a nd a nalyze t he geosmin production u nder d ifferent e nvironments. T he high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) was used to amplify the full-length of geosmin synthase gene from Aphanizomenon gracile (WH-1). Real time PCR (RT-PCR) was applied to quantify the geosmin production in different light and temperature. As a result, geo, a geosmin synthase gene from Aphanizomenon gracile (WH-1) was cloned by hiTAIL-PCR. The full-length open reading frame (ORF) of geo was 2 262 bp, coding for a protein of 753 amino acids. Meanwhile, WH-1 was treated with different environment conditions and mRNA expression levels of geo were quantified by RT-PCR. It was found that low temperature (15 °C), high light intensity (35 μmol m-2 s-1) and continuous light illumination were beneficial to the expression of geo. The successful amplification of geosmin synthase gene verified that hiTAIL-PCR is an effective and simple procedure of low cost. The result provides fundamental knowledge on the monitoring and prevention for odorants.     

Identification and expression of the COI1 gene family in Camellia sinensis and its expression in oolong tea processing北大核心CSCD

茉莉酸受体蛋白COI1(coronatine insensitive 1)是茉莉酸信号转导途径的重要组成部分,为鉴定分析茶树茉莉酸受体COI1基因家族,预测其潜在的分子功能,了解茉莉酸受体COI1基因在乌龙茶加工中应答胁迫的分子机制,利用生物信息学方法对茶树茉莉酸受体COI1进行家族成员鉴定,氨基酸序列、结构域、基因结构、进化分析以及启动子顺式元件分析,结合实时荧光定量分析CsCOI1基因在乌龙茶加工中的表达.结果显示,茶树茉莉酸受体CsCOI1家族有两个成员,均含有F-box和富亮氨酸重复序列(LRRs)两个结构域;单子叶、双子叶的COI1蛋白各聚一支,且与蜜柑进化关系较近;茶树COI1基因家族两个成员均含有3个内含子,启动子顺势元件主要有胁迫响应元件、激素响应元件以及光响应元件;转录组数据说明茶树CsCOI1基因具有较强的组织表达差异性.实时荧光定量分析表明,CsCOI1a基因在室内萎凋后表达显著上调,且15 min、30 min日光萎凋后CsCOI1b基因的表达水平显著上调,同时茉莉酸含量发生显著变化.本研究推测CsCOI1基因可能通过茉莉酸信号转导途径参与乌龙茶加工中萎凋的胁迫响应过程,该结果可为乌龙茶加工品质调控奠定基础.(图8表2参30)     

Identification and expression analysis of the HvENOD93 gene family in barley北大核心CSCD

早期结瘤素93(ENOD93)蛋白在植物根瘤形成初期扮演着重要的角色.基于大麦基因组信息鉴定大麦ENOD93基因家族成员,分析其理化特性、进化关系、基因结构、蛋白质结构和启动子顺式作用元件;并分析ENOD93家族在不同组织和不同基因型(籽粒大小)的表达情况.结果显示,大麦ENOD93基因家族有16个成员,均含有ENOD93基因家族特有的保守结构域;编码区长度在207-627 bp之间,外显子数量有1-4个,平均2.75个,且大部分位于细胞膜上;进化树结果表明与水稻、小麦和玉米等禾本科植物ENOD93基因的亲缘关系较近;启动子顺式元件主要有植物生长发育响应元件、胁迫响应元件以及激素响应元件;大部分HvENOD93基因在灌浆期籽粒和大粒材料中表达量较高.推测大麦HvENOD93基因在籽粒大小形成中起关键性作用;另外,结合其他物种相关基因研究结果,共筛选出3个同源基因.(图4表2参45)     

Isolation,identification, and biological characteristics of a wild Chaga mushroom北大核心CSCD

In this study, a pure culture strain (numbered as F1501) was obtained using tissue separation and purification methods from the sclerotia of Chaga mushroom from Russia. Further researches included studies on classification, optimum growth conditions of mycelia, extracellular polysaccharides from fermentation broth and their antioxidant activity, and artificial acclimation. According to the identification of the internal transcribed spacer (ITS) region, it was confirmed that F1501 was a species of the genus Inonotus and family Hymenochaetaceae, which had 99% similarity with Inonotus obliquus. F1501 was further classified as I. obliquus based on the morphological characteristics of its mycelia and sclerotia. The optimal carbon source, nitrogen source, C/N ratio, growth factor, temperature, and pH value for the growth of the F1501 strain mycelia were maltose, beef extracts, 10/1, B2, 28 °C, and 8.0, respectively. Liquid fermentation of F1501 was performed using PD media and a 10% inoculation amount at 28 °C and 150 r/min for 7 d. The content of polysaccharides in the fermentation broth was 476.32 mg/L, with a total antioxidant activity of 0.19 mmol/L (Trolox) and hydroxyl free radical-scavenging activity of 72.7%. Artificial acclimation study revealed that the fruiting body-like structure was obtained using cottonseed hulls as the main substrate. In the present study, a new strain of I. obliquus and its biological characteristics were explored, which could provide a theoretical basis for the artificial acclimation and development of wild mushrooms. © 2018 Science Press. All rights reserved.     

Isolation,identification, and the biodegradation characteristics of a benzoylurea insecticides-degrading strain北大核心CSCD

A chlorbenzuron, diflubenzuron, and hexaflumuron-degrading bacterium strain M6, was isolated from the activated sludge of an insecticide factory. The strain was identified as Achromobacter sp. according to an analysis on the 16S rRNA gene sequences, morphological, and physiological characteristics. Strain M6 could degrade more than 91% of 100 mg/L chlorbenzuron, diflubenzuron, and hexaflumuron within 48 hours, which could act as the sole carbon source. Strain M6 showed more chlorbenzuron degradation at a temperature range between 25 and 40 ℃ and a pH range between 6.0 and 8.0. The optimal temperature and the initial pH of medium for chlorbenzuron degradation by strain M6 were 30 ℃ and 7.0, respectively; the maximum chlorbenzuron tolerated concentration of strain M6 was as high as 400 mg/L. Strain M6 hydrolyzed 4-acetaminophenol into a purple-red product. Moreover, an approximately 1.4 kb DNA fragment, which could be expressed into an amidase to degrade amide pesticides, was amplified from the genomic DNA of strain M6. The results preliminarily proved that 3 benzoylurea insecticides could be degraded because of strain M6 hydrolyzing their amide bonds. This study obtained a highly efficient degrading strain and provided new resources and valuable information on benzoylurea insecticide degradation. © 2018 Science Press. All rights reserved.     

Screening and degradation characteristics of a tetracycline-degrading bacterial strain北大核心CSCD

Tetracycline is widely used in livestock and poultry breeding industry, which can cause serious problems to the environment. Antibiotic pollution has become an important environmental issue. This study aimed to isolate and identify a well-functioning tetracycline-degrading bacteria strain from activated sludge and to investigate its optimum degradation conditions. The strain was identified through morphological features, Gram staining, and the sequence analysis of 16S rRNA. Furthermore, the temperature, initial pH of the medium, inoculation amount, and type of metallic salt were analyzed to investigate the tetracycline degradation performance of the isolated strain. Based on the single factor test, the method of response surface analysis was adopted to optimize the degradation condition. The strain was named TTC-1 and identified as Klebsiella pneumoniae. The optimum condition for tetracycline degradation was determined as follows: temperature of 34.4 °C, pH of 7.22, and MnSO4 concentration of 0.32 g/L. Under this optimum condition, the predicted tetracycline degradation rate was 93.77%, whereas the observed value was 94.26%. The experimental results showed that the proposed model had high accuracy. TTC-1 showed a good performance in degrading tetracycline, which can provide reference for the bacteria during the biological treatment of tetracycline containing wastewater. © 2018 Science Press. All rights reserved.     

Volatile components and their corresponding synthetic gene expression profile in the fruits of Actinidia chinensis北大核心CSCD

This study aimed to identify the difference in volatile aromatic components and the relationship with the expression pattern of their corresponding bio-synthesis genes in six kiwifruit (Actinidia chinensis) varieties (Cuiyu, Jintao, Jinyan, Chuhong, Donghong, and Xixuan). To provide a foundation for kiwifruit variety recognition, fruit quality evaluation, and molecular-assisted breeding, the volatile aromatic components in the ripe fruits of six kiwifruit varieties were evaluated by head space-solid phase micro extraction/gas chromatography-mass spectrometry. The aroma-synthesis-related genes, including acyltransferases (AcAT16), lipoxygenase (AcLox2), and terpene synthase genes (AcTPS1), were detected by the real time-quantitative polymerase chain reaction (qPCR) during the postharvest stage of fruits. Ninety-two aroma chemicals were identified in the tested kiwifruit cultivars. There were 35, 32, 30, 44, 28, and 17 of aromatic compounds in Cuiyu, Jintao, Jinyan, Chuhong, Donghong, and Xixuan, respectively. Esters were the main aroma components in Cuiyu, Jintao, and Jinyan. The major aromatic compounds of Chuhong, Donghong, and Xixuan were aldehydes and terpenoids. The expression level of AcATs16 and AcLox2 increased, and then decreased during the ripening of kiwifruit fruits. The expression of AcATs16 was significantly higher in Cuiyu and Jinyan than in other varieties. AcLox2 indicated significant abundance in Cuiyu and Chuhong. AcTPS1 was up-regulated in Jintao, Donghong, and Xixuan with fruit ripening; however, this was not observed in Cuiyu, Chuhong, and Jinyan. The difference in the composition and content of volatile aromatic components contributes to the difference in aroma in different kiwifruit cultivars. The diverse expression of AcATs16, AcLox2, and AcTPS1 might be closely related to the synthesis of ethyl butyrate, (E)-2-hexenal, and eucalyptol, respectively. © 2018 Science Press. All rights reserved.     

Genome-wide identification and hormone expression patterns of the MaPIF family of banana genes北大核心CSCD

植物光敏色素作用因子(phytochrome interacting factor,PIF)是广泛分布于植物体内的一种转录因子,在植物的生长发育方面有着重要的作用.基于香蕉基因组数据,对香蕉MaPIF基因家族进行基因组鉴定,采用生物信息学分析方法对其进行命名,分析理化性质、蛋白质结构、基因结构、启动子顺势作用元件以及构建系统进化树;分析PIF家族在不同激素处理下的表达情况.结果显示,香蕉MaPIF家族有7个成员,均含有高度保守的bHLH结构域;编码区长度在1 116-2001bp之间,至少包含5个内含子,且大部分位于细胞外;进化树结果可以发现与拟南芥、水稻以及玉米PIF的亲缘关系较近;顺式作用元件预测结果显示,MaPIF上存在多种与激素和光相关的响应元件.qRT-PCR结果显示,MaPIF3-1、MaPIF4、MaPIF4-1在生长素(IAA)、赤霉素(GA)、生长素抑制剂(NPA)处理中均有显著表达,除此之外,所有成员在脱落酸(ABA)处理下均有明显表达.本研究表明MaPIF在香蕉生长发育中激素调控有重要作用.(图7表3参45)     

Isolation,identification, culture conditions,and laccase production of a wild mushroom 'Huaier'北大核心CSCD

In the present study, fruiting bodies of a wild medicinal mushroom, 'Huaier, ' were collected from Populus canadensis in the Beijing Xiangshan Park. The pure culture strain was obtained from fruiting bodies using the tissue isolation method. It was stored and numbered as XS-01. It was systematically classified using morphological and ITS identification. Further studies were focused on mycelia optimum culture conditions and laccase production by liquid fermentation. A 598-bp partial ITS region sequence (GenBank accession number KY93348) was obtained using PCR method. Phylogenetic tree and genetic distance analysis were performed using the MEGA 6.0 software. The present strain possessed the highest homology (100%) with Perenniporia robiniophila, and the genetic distances were 0.000. Based on the ITS sequencing and morphological characteristics of fruiting bodies and mycelia, XS-01 was identified as P. robiniophila. Based on mycelial growth rate and quality, mycelia optimum culture conditions were revealed to be as follows: the optimum carbon sources were starch and maltose, the optimum nitrogen source was yeast extracts, the optimum C/N ratio range was 30/1 - 60/1, the best growth temperature was 32 °C, the optimum pH was 7, and the optimum growth factor was VB1. Further study of Cu2+ on extracellular laccase production revealed that 1.0 mmol/L Cu2+ could significantly enhance the enzyme production, with the highest activity of 417.5 U/mL when cultured for 96 h and an increase ratio of 93.4% to the control. On the other hand, 2.0 mmol/L Cu2+ can markedly decrease the enzyme production laccase activity at 96 h to 79.0 U/mL, which was 36.6% of that of the control. In conclusion, a pure strain of T. robiniophila with high extracellular laccase activity was obtained, suggesting its potential application for artificial cultivation and laccase production. © 2018 Science Press. All rights reserved.     

Isolation,identification, and the degradation characteristics of a BDE-47 degrading strain北大核心CSCD

In an effort to remove BDE-47 residues from the environment, a bacterial strain that is capable of utilizing BDE-47 as the sole carbon source was isolated and screened from soil collected from an e-waste recycling area in Tianjin to analyze the degradation properties. The strain was preliminarily identified as Enterobacter sp. according to a 16S rDNA gene sequence analysis. The strain degraded 35.8% of 525 μg/L of BDE-47 in 35 d when the initial concentration of bacteria was 7.1 × 105 cells/ mL. The product of the biodegradation of BDE-47 was BDE-28. The biodegradation of BDE-47 fit well with first-order kinetics, and its degradation kinetics was ln Ct = - 0.104t + 6.22. With the addition of an electron acceptor, such as Fe3+, SO4 2- and NO3 -, the BDE-47 degradation rate was significantly increased to 49.8%, 59.1%, and 67.3%, respectively. The above results revealed that the strain could degrade BDE-47, which is of importance in the application of environmental bioremediation of BDE-47. © 2018 Science Press. All rights reserved.     

Screening and characterization of extremophiles in spacecraft assembly environment北大核心CSCD

Spacecrafts need to strictly control microorganisms before entering space flight. The Spacecraft Assembly, Integration, and Test Center (AITC) is an important environmental source for spacecrafts to carry microorganisms. The assembly clean room has characteristics of ventilation, dryness, and lack of nutrients that are not conducive to the growth and reproduction of microorganisms, except for extremophiles. In this study, based on the internal air and surface environment of the AITC in China, 13 strains of bacteria were identified by plate culture and 16S sequence analysis, and their extreme environmental tolerance, antibiotic resistance, and film-forming ability were tested. All these bacteria belonged to the Firmicutes and Proteobacteria phyla, and nine strains belonged to the Bacillus genus. All 13 strains of bacteria were salt-tolerant, acid-tolerant, and alkali-tolerant, and 69.2% of the bacteria survived heat shock at 80 ℃. Among these, one strain of Sphingomonas sp. JCM7513 isolated from the surface environment was insensitive to all the tested antibiotics and had strong drug resistance, while the other 12 strains were sensitive to most β-lactam antibiotics but had strong resistance to tetracyclic antibiotics and erythromycin. Most of the isolated bacteria exhibited strong biofilm-forming abilities. The study showed that there are a certain number of extremophiles in the spacecraft assembly environment. To protect spacecrafts from biological corrosion and planetary protection forward pollution, more effective monitoring and elimination methods are needed. © 2022 Science Press. All rights reserved.     

Assessment of heavy metal pollution and health risks in a main river in Huayuan County,Xiangxi, Hunan Province北大核心CSCD

To study heavy metal pollution and assess the health risk of river water in Huayuan County, Xiangxi, Hunan Province, 11 water samples were collected from the Huayuan River and Brother Rivers in August and December 2016. Heavy metal (Pb, Zn, Cr, Cu, Fe, and Ni) concentrations were determined from the samples. The health risk assessment model recommended by the U.S. Environmental Protection Agency (USEPA) was applied to assess the health risk of heavy metals in the main surface waters of Huayuan County. The results indicated that the concentrations of heavy metals (Pb, Zn, Cr, Cu, Fe, and Ni) of surface water in the research area were 2.57 × 10-3, 4.66 × 10-4, 1.65 × 10-3, 6.27 × 10-4, 0.19, and 8.50 × 10-4 mg/L, respectively. The health risk of surface waters with heavy metals was high. Therefore, the chemical carcinogenic substance (Cr) health risk index was five or six times higher than that of chemical non-carcinogens (Pb, Zn, Cu, and Ni). The average health risk indices of non-carcinogenic substances were in the order Pb > Cu > Zn > Ni. The correlation and principal component analysis of surface water showed that the six heavy metal elements were composed of three main components in the main surface waters of the county. The first principal component was comprised of Fe and Ni (33.28%), which was mainly from internal pollution. The second component was comprised of Cu and Cr (26.98%), which was primarily due to industrial waste water, rainwater leaching mineral waste produced by heavy metal mining, and smelting enterprises. The third component, resulting from geochemical pollution, was Zn (17.10%). The health risk indices triggered by heavy metal in surface waters was high. Heavy metal pollutants in the research area need to be controlled in the order Cr, Pb, Cu, Zn and Ni. © 2018 Science Press. All rights reserved.