袋料香菇菌株ITS及β-tubulin序列分析

以香菇栽培袋分离菌株为研究材料,以香菇931-9为对照菌株,采用ITS及β-tubulin序列分析方法对研究材料进行鉴定。结果表明,香菇栽培袋是2种香菇菌株菌丝的混杂,与对照菌株931-9不是同一个菌株,证实青川县香菇栽培袋分离菌株与对照菌株931-9不是同一菌株。     

棉花β-tubulin基因家族的鉴定及其在纤维发育中的表达

【目的】β-微管蛋白是棉纤维细胞形态建成的基本结构单位,在纤维发育过程中具有重要作用。通过鉴定棉花β-tubulin基因家族成员,并进行生物信息学和表达模式分析,为深入探究β-tubulin基因在棉花纤维发育中的作用提供理论依据。【方法】利用BLAST方法,在4个棉种基因组中鉴定β-tubulin基因家族成员,并结合ProtParam tool分析理化性质、MEGA7.0构建系统进化树、Mapchart2.2绘制染色体定位图、MEME分析保守基序、PlantCARE分析启动子顺式作用元件;根据39个材料发育纤维转录组数据分析陆地棉β-tubulin基因家族的表达水平,并利用Spearman相关性分析鉴定影响纤维品质性状形成的β-tubulin基因。【结果】在陆地棉（Gossypium hirsutum,AD1）、海岛棉（Gossypium barbadense,AD2）、亚洲棉（Gossypium arboretum,A2）和雷蒙德氏棉（Gossypium raimondii,D5）基因组中分别鉴定到36、37、19和18个β-tubulin基因,且在四倍体棉种中的数目约是二倍体棉种数...     

新疆南部塔河流域柳树腐烂病病原菌的鉴定

【目的】通过对病原菌形态学和培养形状观察,结合分子生物技术ITS和β-tubulin序列分析,明确新疆南疆柳树腐烂病菌种类和遗传分化。【方法】采集柳树腐烂病并获得病菌纯培养,对腐烂病菌分生孢子器进行纵横切片并观察记录形态数据,将病原菌置于不同营养条件和环境条件下观察记录其培养形状,采用ITS和β-tubulin序列分析方法结合,构建系统发育树。【结果】分子孢子器和分子孢子形态特征与金黄壳囊孢菌(C.chrysosperma Per.ex Fr.)一致;菌丝生长最适温度20~30℃,最适pH值为5~6,最适碳氮源分别是葡萄糖和蛋白胨;经β-tubulin序列分析,与壳囊孢属不同种类的序列相似度为96%以上;在室内离体接种条件下病原菌均能成功侵染其他5种树木。【结论】引起新疆南部塔河流域柳树腐烂病原菌为金黄壳囊孢菌(C.chrysosperma per.ex Fr),不同来源的病原菌存在一定的遗传分化,在室内可侵染杨树、胡杨、枣树、梨和苹果,并可在胡杨和杨树上产生分生孢子器。     

美洲斑潜蝇不同寄主种群及地理种群间的β-tubulin基因序列分析

 【目的】通过对美洲斑潜蝇β-tubulin基因序列的比较,分析不同寄主种群和地理种群的遗传分化情况。【方法】对中国美洲斑潜蝇Liriomyza sativae Blanchard 5个寄主种群以及6个地理种群的β-微管蛋白基因（β-tubulin gene）进行测序,运用软件DNAStar和MEGA对美洲斑潜蝇不同地理种群及寄主种群的β-tubulin基因序列的遗传分歧及相似性进行分析,并建立系统发生树。【结果】DNAStar和MEGA两种软件对美洲斑潜蝇不同寄主种群以及地理种群间的遗传关系的分析可得到相似的结果。在美洲斑潜蝇寄主种群和地理种群序列变异分析中,共发现8个变异位点,序列长度比较保守,均没有碱基的插入或缺失。美洲斑潜蝇不同寄主种群之间以及地理种群之间的β-tubulin基因序列相似性极高,都在98%以上。【结论】虽然基于β-tubulin基因序列所显示的美洲斑潜蝇各寄主种群之间以及地理种群之间的遗传分化很小,但是其分化趋势分别与对寄主的嗜好程度以及地理分布基本吻合。     

1株侵染蓖麻的葡萄座腔菌菌株鉴定

从江苏盐城地区种植的蓖麻上采集分离到1株编号为YDJ08的病原菌。通过形态学观察和分子生物学研究对其进行鉴定,结果显示:在PDA培养基上,真菌菌落形态为圆形,菌落初期为白色,后转变为黑色,分生孢子呈梭形、薄壁、外壁光滑、无色;病原菌在蓖麻植株上接种后表现为木质部变黑;ITS和β-tubulin序列分析表明该菌株与Gen Bank中葡萄座腔菌(Botryosphaeria dothidea)的序列相似性分别高达100%和99%。综合形态学和序列比对分析推断该菌株为葡萄座腔菌。这是首次报道侵染蓖麻的葡萄座腔菌,该菌株的ITS和β-tubulin序列的Gen Bank登录号为KJ530706、KJ530707。     

不同分子指标鉴定四川雅江鸡蛋菌的差异

以采自四川雅江的一株野生鸡蛋菌作为研究材料,利用CTAB法提取基因组DNA,然后利用PCR技术分别扩增ITS、LSU、RPB2、β-tubulin和EFI-α等DNA序列相应区段,并连接到T载体上进行DNA测序;利用GenBank核酸数据库对测序得到的不同序列进行同源比对,并从GenBank检索获取相似序列,与测序得到的不同序列分别构建系统发育树。结果揭示,以ITS和LSU序列分析鉴定该野生菌为肉桂鹅膏菌(Amanita cinnamomescens),而以RPB2、β-tubulin和EFI-α序列分析鉴定结果为红黄鹅膏黄褐变种(Amanita hemibapha var.ochracea);最后综合ITS、LSU、RPB2、β-tubulin和EFI-α序列分析和结合形态学观察,将四川雅江鸡蛋菌鉴定为红黄鹅膏黄褐变种(Amanita hemibapha var. ochracea)。在鸡蛋菌的分类鉴定中应考虑更多的分子指标,不能以单一的ITS和LSU序列作为单一的标准。     

一株脱氧雪腐镰刀菌烯醇转化菌的分离、鉴定及其转化效果研究

以脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)为唯一碳源,通过富集培养从土壤中分离到一株可以转化DON的真菌,命名为NJA-1.用引物Bt2a、Bt2b扩增了该菌的β-tubulin基因序列,GenBank的登录号为DQ902579.通过基因序列分析和形态学观察最终鉴定该菌株为塔宾曲霉(Aspergillus tubingensis).该菌与塔宾曲霉AY820009的同源性为99%.在pH 60、30 ℃、14 d内其对DON的平均转化率为944%.HPLC-MS分析结果表明,DON转化产物的相对分子质量为3144,比DON的相对分子质量(2963)增加了181.该菌在无机盐培养基中对DON的最高耐受浓度为40 mg·L-1.提示:该菌可用于DON的生物转化.     

江西省丝瓜果实腐烂病病原及寄主范围研究

为明确江西省丝瓜果实腐烂病病原菌种类及病原菌寄主范围,从江西省南昌市和赣州市丝瓜种植区采集具有典型症状的腐烂病病果并进行病原菌分离,采用形态学观察、致病性测定和分子生物学技术对其种类进行鉴定,并接种7种作物幼苗以明确其寄主范围.结果表明,从丝瓜烂果病病样中共分离到2株菌株,分别命名为SG-1和SG-2,2株菌株形态特征...     

缺氧海马神经元中KATP对p53下游基因α-tubulin表达的影响

目的了解缺氧海马神经元中,KATP对p53下游基因α-tubulin表达的影响。方法原代培养7d后,将神经元随机分为常氧组、常氧＋药物（KATP激动剂二氮嗪和抑制剂甲苯磺丁脲）组、单纯缺氧组（5%CO2,和95%N2）、缺氧＋药物（KATP激动剂二氮嗪和抑制剂甲苯磺丁脲）组,采用倒置显微镜和NF免疫组织化学法观察细胞生长和形态,MTT法检测细胞凋亡率,RT-PCR检测各下游基因的mRNA表达水平。结果单纯缺氧组、缺氧＋二氮嗪组和缺氧＋甲苯磺丁脲组的多级神经元的百分率分别是（20.6±2.0）%、（26.1±1.7）%和（8.7±1.7）%,各加药组与单纯缺氧组比较均有统计学意义（P＜0.01）;细胞存活率分别是（75.7±2.8）%、（55.7±2.5）%和（81.1±2.4）%,各缺氧＋药物组与单纯缺氧组比较差异有统计学意义（P＜0.01）。缺氧组中α-tubulin的mRNA表达水平与常氧时比较明显下降（P＜0.01）,缺氧＋二氮嗪组和缺氧＋甲苯磺丁脲组的mRNA表达水平与单纯缺氧组相比差异无统计学意义（P＞0.05）。而在常氧时各用药组中多极神经元的百分率、细胞存活率以及α-tubulin的mRNA表达水平与常氧组相比差异没有统计学意义。结论缺氧时KATP对细胞具有保护作用,KATP对α-tubulin的表达没有显著性影响。     

Inheritance and Expression of Potato Proteinase Inhibitor Gene Ⅱ (pinⅡ) in Transgenic Rice

The inheritance and expression of bar gene and pinⅡ gene were studied in three transgenic ricelines and their F2 hybrid populations, which were created through hybridization with a PGMS line, ZAU11S.By Basta painting, PCR analysis and determining of the inhibitory trypsin activity, the results show that bargene and pinⅡ gene in rice F2 population fit the simple Mendel's low of inheritance and close linkage, but afew plants in F2 have not sufficiently expressed. The wound inducible pin Ⅱ gene has an expression regulatedspatially and temporally, and the signal transduction pathway is not only upward, but also downward. The in-ducible expression of pinⅡ in different rice transgenic lines is not completely coincident.     

The Sequence Variations of Intron-3 of the α-Amylase Gene in Adzuki Bean

This study describes variation of intron-3 of a-amylase gene from 156 breeds of adzuki beansusing SSCP(single-strand conformation polymorphism)analysis. Based on a-amylase gene structure and se-quence, A pair of PCR primers, F (CCTACATTCTAACACACCCT) and R (GCATATTGTGCCAGTACAAT)were designed to amplify intron-3 fragments of a-amylase gene. 14 variant types were detected, including 13,9, 10, 4 variant types in the wild, weed, locally cultivated and modern brought-up adzuki beans respectively,9, 8, 7 variant types of the wild adzuki beans from Japan, China and Korea respectively, and some other va-riant types in the local adzuki beans from China and Bhutan. 60 % of subjects of cultivated races were found tobe EE type in the experiment. In addition, sequence analysis of intron-3 of α-amylase gene from 8 varianttypes reveals the evolution process of various variant types in adzuki beans.     

Construction and Expression of Methionine-rich and Lysine-rich Fusion Gene inBacillus natto

Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed directly affect livestock protein synthesis. Bacillus natto has excellent probiotic properties. In this experiment, we used the genetic engineering method, fusion PCR technique, to connect methionine-rich gene(zein) from maize endosperm protein with lysine-rich gene(Cflr) from the pepper anther, then the fusion gene was inserted into the expression vector p HT43, and the recombinant plasmid p HT43/zein-Cflr was constructed. The recombinant plasmid was transferred into Bacillus natto, and induced by IPTG for the expression of the fusion gene. We found an apparent band at 40 ku site for the recombinant strain by SDS-PAGE. The contents of methionine and lysine were individually detected with HPLC, the quantities of methionine and lysine in the recombinant strain increased by 18.37% and 24.68% than the wild one, respectively. We also verified the stability of the recombinant bacterium during passaging, and found the stability was 100%. This study provided research-basis for the application of the recombined Bacillus natto as feed additive.     

Postulation of Leaf Rust Resistance Genes in Seven Chinese Spring Wheat Cultivars

To detect the leaf rust resistance genes in the 7 Chinese spring wheat clultivars Shenmian 99025, Shenmia 99042, Shenmian 85, Shenmian 91, Shenmian 96, Shenmian 1167 and Shenmian 962, Thatcher, Thatcher backgrounded near-isogenic lines and 15 pathotypes of P. triticina were used for gene postulate at the seedling stage, and 9 of the 15 pathotypes were used in the field tests. Molecular markers closely linked to, or co-segregated with resistance genes Lr1, Lr9, Lr10, Lr19, Lr20, Lr21, Lr24, Lr26, Lr28, Lr29, Lr32, Lr34, Lr35, Lr37, Lr38, and Lr47 were screened to assist detection of the resistance genes. As results, 4 known resistance genes, including Lr1, Lr9, Lr26, and Lr34, and other unknown resistance genes were postulated singly or in combination in the tested cultivars. Shenmian 85, Shenmian 91, Shenmian 96, Shenmian 962, Shenmian 1167, and Shenmian 99042 are potentially useful for wheat production and breeding programs. The result suggested that combining gene postulation, molecular markers and pedigrees is effective and more accuracy method to know the resistance genes in cultivars.     

Molecular Phylogenetic Analysis of the Main Lineages of Nymphalinae (Nymphalidae: Lepidoptera) Based on the Partial Mitochondrial COI Gene

The phylogenetic relationships of the subfamily Nymphalinae (sensu Chou 1994) were analyzed based on 1488bp of mtDNA cytochrome oxidase subunit I (COI) gene sequence data obtained from 24 individuals, along with those of eight species obtained from GenBank. The base compositions of this COI fragment varied among the individuals as follows: T 39.9%, C 14.6%, A 32.2%, and G 13.4%, with a strong AT bias (72.1%), as usually found in insect mitochondrial genomes. The A T contents of the third, second, and first codon positions of the COI fragments in this study was 92.4, 62.2, and 61.4%, respectively. The phylogenetic trees were reconstructed by neighbor-joining (NJ), maximum likelihood (ML), and Bayesian methods by using Byblia anvatara as outgroup. Phylogenetic analyses based on the COI gene sequence data created very similar topologies, which were producing trees with two main clades A and B, and five subclades. The data indicated that the tribes Nymphalini and Hypolimni (sensu Chou 1994) are not monophyletic groups, and the genus Junonia should be removed from Nymphalini to Hypolimni (=Junoniini). On the basis of the data, the Symbrenthia and Araschnia had a relative distant relationship with the rest of Nymphalini. The relationships of species in the Nymphalini were confirmed via the NJ, ML, and Bayesian methods, namely ((((Nymphalis Kaniska) Polygonia) Aglais) Vanessa) (Symbrenthia Araschnia). This investigation provides a little novel information for Chinese researches of butterflies.     

Screening and characterization of a novel ruminal cellulase gene(Umcel-1) from a metagenomic library of gayal(Bos frontalis)

Gayal is a rare semi-wild bovine species found in the Indo-China.They can graze grasses,including bamboo leaves,as well as reeds and other plant species,and grow to higher mature live weights than Yunnan Yellow cattle maintained in similar harsh environments.The aim of this study was to identify specific cellulase in the gayal rumen.A metagenomic fosmid library was constructed using genomic DNA isolated from the ruminal contents of four adult gayals.This library contained38400 clones with an average insert size of 35.5 kb.The Umcel-1 gene was isolated from this library.Investigation of the cellulase activity of 24 random clones led to the identification of the Umcel-1 gene,which exhibited the most potent cellulase activity.Sequencing the Umcel-1 gene revealed that it contained an open reading frame of 942 base pairs that encoded a product of 313 amino acids.The putative gene Umcel-1 product belonged to the glycosyl hydrolase family 5 and showed the highest homology to the cellulase（GenBank accession no.YP₀04310852.1）from Clostridium lentocellum DSM 5427,with 44%identity and 62%similarity.The Umcel-1 gene was heterologously expressed in Escherichia coli BL21,and recombinant Umcel-1 was purified.The activity of purified recombinant Umcel-1 was assessed,and the results revealed that it hydrolyzed carboxymethyl cellulose with optimal activity at pH 5.5 and 45℃.To our knowledge,this study provides the first evidence for a cellulase produced by bacteria in gayal rumen.     

Bacillus subtilis Co-transfected with a Lysine-rich and a Methionine-rich Protein Gene and Its Effect on Cow Milk Production

The probiotic Bacillus subtilis(B. subtilis) was widely applied in animal production as feed additive. Lysine(Lys) and methionine(Met) were the two most important limiting amino acids in livestock animal feed. Raising Lys and Met contents in B. subtilis would provide better effects for animal production and save Lys and Met supplements. We still didn't know whether Lysrich and Met-rich protein genes from plants could be transfected into B. subtilis and expressed at a high level so as to improve animal production, such as milk production as an additional diet. The Lys-rich protein gene(Cflr) and Met-rich 10 ku-δ Zein were cloned from pepper anther and maize endosperm, respectively. Then they were constructed into plasmids individually and successfully cotransfected into B. subtilis. Upon IPTG induction, m RNAs and protein expressions could be observed. Lys and Met contents in the fermentation broth were raised by 65.92% and 46.39%, respectively. After feeding 200 g and 400 g · cow~(-1) · d~(-1), transgenic B. subtilis fermentation broth, the milk yield, milk protein and milk fat contents all significantly increased. The Lys-rich protein gene(Cflr) and Met-rich 10 ku-δ Zein were successfully transfected into B. subtilis. Contents of Lys and Met in the transgenic B. subtilis obviously raised and the fermentation broth of the transgenic bacteria could effectively improve milk yield and quality.     

The Construction of the Probe for Amylase n Gene Cloning from Bacillus halodurans Strain 38C1-1

Primers and probes were established according to the sequences of the alpha-amylase genes of Bacillus, halodurans C-125, Thermus sp. IM6501, B. stearothermophilus ET-1, and B. acidopullulytics. Primers were designed and a 0.2 kb DNA fragment was amplified, the fragment was successfully used for the detection of the amylase Ⅱ gene in a 2 842 bp region from Bacillus halodurans strain 38C1-1.     

番茄部分WRKY基因非生物胁迫表达和SlWRKY50基因沉默分析

前期研究已完成番茄WRKY转录因子家族分析,发现番茄WRKYⅡa和Ⅱb亚族基因多与抗逆相关,因此文章选取6个WRKYⅡa和Ⅱb亚族基因Sl WRKY13、Sl WRKY24、Sl WRKY31、Sl WRKY50、Sl WRKY62、Sl WRKY63,运用q RT-PCR分析方法,分析逆境胁迫下表达模式。结果表明,Sl WRKY24干旱、盐、低温胁迫下表达受抑制,另外5个基因在三种胁迫处理中,除干旱胁迫处理Sl WRKY50基因表达量下降,其余均不同程度上调表达。低温胁迫处理下Sl WRKY50基因表达量与对照相比表现极显著差异。Sl WRKY50基因沉默分析结果表明,Sl WRKY50基因沉默后,下调Sl WRKY50基因表达,干旱、高盐胁迫下植株叶片Pro、SOD、MAD含量水平与对照相比变化较小,低温胁迫下番茄植株叶片Pro和SOD含量低于对照,MAD提高含量,植株对低温逆境胁迫耐受能力下降。研究为进一步探讨WRKY基因家族功能提供参考及理论依据。     

Lentivirus Mediated Gene Manipulation in Trophectoderm of Porcine Embryos

Development of tools that can manipulate gene expression specifically and efficiently in the trophectoderm（TE） lineage would greatly aid understanding the roles of different genetic pathways in TE versus embryonic lineages. Here, we showed first time that short-term lentivirus infection of porcine blastocysts could lead to rapid expression of transgene specifically in TE cells. Efficient TE-specific gene knockdown could also be achieved by lentivirus-mediated pol III-driven short hairpin RNA（shRNA） and TE-specific gene expression could be temporal controlled efficiently by combining this system with Tet-On system. This lentivirus lineage-specific infection system would facilitate gene function studies in porcine pre-implatation embryos by specifically knockdown or overexpression of these genes in TE.