Genetic Variations Among Liriomyza sativae Blanchard Populations Indicated by β-tubulin Gene Sequences

To analyze the genetic differentiation of the host- and geo-populations of Liriomyza sativae Blanchard, the β-tubulin gene of 5 host-populations and 6 geo-populations of L. sativae was sequenced. Subsequently, the sequences were analyzed by biosoftware DNAStar and MEGA, and the phylogenetic trees constructed. The results obtained by the two softwares were similar, that is, the sequences of β-tubulin gene were more than 98% homologous, among the host- and geo-populations of L. sativae, with only 8 variable sites, but no insertions and deletions were detected. It seems that differentiations in β-tubulin gene among the host- and geo-populations of L. sativae are related to the hobby to hosts and the geographical distributions, respectively.     

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.     

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.     

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.     

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.     

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。     

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.     

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.     

牛Myostatin基因单核苷酸多态性分析

Myostatin基因即肌肉生长抑制素,是一种肌肉生长的负调控因子。应运PCR-SSCP和测序的方法对中国秦川牛、南阳牛以及国外引入品种皮埃蒙特牛双肌基因的第三外显子进行了多态性分析。结果表明:第三外显子938处G→A的单核苷酸的突变造成了南阳牛、皮埃蒙特牛第三外显子扩增片段多态性,秦川牛则不然。     

马MxA基因第13外显子的多态性研究(英文)

[Objective] To investigate the polymorphism of the thirteenth exon of MxA gene in 4 species of horse. [Method] The thirteenth exon of MxA gene fragments were amplified from genomic DNA of Sanhe horse, Xinihe horse, Wushen horse and Baerhu horse with the primers designed according to the MxA sequence announced in GenBank; the polymorphism of MxA gene was detected by PCR-SSCP and the products were sequenced. [Result] The polymorphism of the thirteenth exon of MxA gene appeared only in Wushen horse, the 2 081 nt of which mutated from guanine (G) to adenine (A) and the corresponding amino acid of which changed from glutamate (Glu) to alanine (Ala). [Conclusion] The study provided a basis for exploring the antiviral effect of MxA protein.     

芒果精氨酸脱羧酶基因MiADC的克隆及表达分析（英文）

精氨酸脱羧酶(Arginine decarboxylase,ADC)在植物多胺生物合成中发挥着重要作用。为了研究精氨酸脱羧酶基因(ADC)在芒果中的结构与表达情况,试验采用RACE技术从芒果嫩叶中克隆获得芒果精氨酸脱羧酶基因MiADC,并以MiADC基因的cDNA序列为基础,采用qRT-PCR分析该基因的表达情况。序列分析结果显示:芒果MiADC基因全长3 089 bp,包括575 bp 5’-UTR,2 178bpORF,311bp3’-UTR和25bppoly(A),且无内含子。NCBI比对分析显示:该基因与枳(Poncirus trifoliate)精氨酸脱羧酶基因(HQ008237.1)的相似性最高,为78%;其预测蛋白同样与枳(Poncirus trifoliate)精氨酸脱羧酶(AEE99192.1)的相似性最高,为78%。系统发生树分析表明:芒果MiADC与枳(Poncirus trifoliate)的精氨酸脱羧酶处于同一进化枝上。表达分析显示:MiADC基因在根、茎、叶、花、幼果、成熟果组织中均有表达,且该基因的转录水平受低温处理的影响。因此,芒果MiADC基因可能是一个低温胁迫应答基因。     

不同品种猪生长激素基因的PCR-RFLPs分析

采用PCR-RFLPs技术,对香猪、上海白猪、大约克夏猪生长激素基因-119～＋715区域共834bp片段的扩增产物分别用ApaI、DraI、MspI三种限制性内切酶检测酶切位点的多态性。结果显示：ApaI酶切时．三个品种的猪都产生了3种基因型（AA、BB和AB）,BB基因型的频率以大约克夏猪最高．AB基因型的频率以香猪最高．三个猪种间基因型频率、等位基因频率的差异不显著（P〉0．05）;DraI酶切时．各品种猪均未呈现酶切位点的多态性;MspI酶切时,仅大约克夏猪表现出2种基因型（CC、CD）,香猪、上海白猪未产生酶切位点多态性。结论：AB基因型的频率以香猪为最高,它可能是小型猪的有利基因型;在香猪和上海白猪中DraI和Mspl酶切位点的多态性比较贫乏。     

蛋氨酸铬对鲤糖代谢相关酶活性及IR、GLUT2和SGLT基因表达的影响

为探究蛋氨酸铬(CrMet)对鲤Cyprinus carpio糖代谢相关酶活性及糖代谢相关基因表达的影响,以酪蛋白为蛋白源,豆油为脂肪源,配制7组纯化饲料,其中Cr~(3+)水平分别为0(对照)、0.1、0.2、0.4、0.8、1.6、3.2 mg/kg。选取初始体质量为(40.95±4.80)g的鲤,随机分为7组,分别投喂7种饲料,每个组设置3个重复,每个重复放60尾鱼,饲养8周后,检测其肝胰脏糖代谢相关酶活性;禁食48 h后再投喂,再投喂0、3、6、12、24、48 h时检测Cr~(3+)水平为0(对照)、0.8、3.2 mg/kg时鱼肝胰脏IR、GLUT2和肠道SGLT基因的表达量。结果表明:添加0.8 mg/kg Cr~(3+)组的组己糖激酶(HK)、丙酮酸激酶(PK)、磷酸果糖激酶(PFK)和琥珀酸脱氢酶(SDH)活力均显著高于对照组(P0.05),磷酸酵式丙酮酸激酶(PEPCK)活力显著低于对照组(P0.05);而添加Cr~(3+)并未对葡萄糖-6-磷酸脱氢酶(G6PDH)活力产生显著影响(P0.05);投喂24、48 h时,0.8 mg/kg Cr~(3+)组IR mRNA表达量显著高于0、3.2mg/kg Cr~(3+)组(P0.05);投喂3 h时,0.8、3.2 mg/kg Cr~(3+)组GLUT2 mRNA表达量均显著高于对照组(P0.05),12、24、48 h时,0.8 mg/kg Cr~(3+)组GLUT2 mRNA表达量显著高于0、3.2 mg/kg Cr~(3+)组(P0.05);而不同Cr~(3+)添加水平对鲤肠道SGLT mRNA表达量无显著性影响(P0.05)。研究表明,在饲料中添加CrMet能够提高鲤对糖的利用能力,建议Cr~(3+)添加水平为0.8 mg/kg。     

Genome-wide analysis of the calcium-dependent protein kinase gene family inGossypium raimondii

Plant calcium-dependent protein kinases(CDPKs) play important roles in diverse physiological processes by regulating the downstream components of calcium signaling. To date, only a few species of the plant CDPK gene family have been functionally identified. In addition, there has been no systematic analysis of the CDPK family in cotton. Here, 41 putative cotton CDPK(Gr CDPK) genes were identified via bioinformatics analysis of the entire genome of Gossypium raimondii and were classified into four groups based on evolutionary relatedness. Gene structure analysis indicated that most of these Gr CDPK genes share a similar intron-exon structure(7 or 8 exons), strongly supporting their close evolutionary relationships. Chromosomal distributions and phylogenetics analysis showed that 13 pairs of Gr CDPK genes arose via segmental duplication events. Furthermore, using microarray data of upland cotton(G. hirsutum L.), comparative profiles analysis of these Gh CDPKs indicated that some of the encoding genes might be involved in the responses to multiple abiotic stresses and play important regulatory roles during cotton fiber development. This study is the first genome-wide analysis of the CDPK family in cotton, and it will provide valuable information for the further functional characterization of cotton CDPK genes.     

Expression of a Lysine-rich Gene in Bacillus subtilis 168

Lysine-rich protein gene (lys) was cloned from winged bean (Psophocarpus tetragonolobus (L.) DC), and cloned into prokaryotic expression vector pHT43, the recombinant plasmid pHT43/lys were constructed and then transferred into Bacillus subtilis168, upon IPTG induction, the recombinant protein was expressed, and the content of lysine was detected by HPLC. The result showed that lysine content increased by 9.85%. It was suggested that introducing lys gene into Bacillus subtilis 168 was an effective way to improve its nutrition quality.