大豆铁蛋白cDNA的克隆及植物表达载体的构建

以大豆为材料,利用RT-PCR法扩增得到大小约750bp的片段,将这个片段连接到克隆载体pBlueskript SK＋上进行测序,结果证明,此片段就是大豆的铁蛋白基因。将此片段再正向插入到植物表达载体pBI121的CaMV35s启动子和NOS终止子之间,构建了植物表达载体pBIFe,并成功地将该载体导入根癌农杆菌EHA105。此项工作为获得表达铁蛋白的转基因植物奠定了基础。     

丛枝菌根真菌和糖醇螯合钙对草莓采后果实硬度和细胞壁酶活及其关键基因表达的影响

以盆栽‘红颜’草莓为试材,研究了摩西管柄囊霉（Funneliformis mosseae）和糖醇螯合钙不同浓度（0.10%、0.15%、0.20%）处理对草莓采后果实硬度和细胞壁酶活的变化,同时,初步探究了不同处理对草莓果实软化关键基因FaPG1、FaβGal4的表达水平的影响。结果表明,与对照相比,以糖醇螯合钙单独施用（0.15%和0.20%）、接种丛枝菌根真菌（Arbuscular mycorrhizal fungi, AMF）配施糖醇螯合钙均能显著提高草莓果实硬度。同时,不同处理均抑制多聚半乳糖醛酸酶（PG）、果胶甲酯酶（PME）、β-半乳糖苷酶（β-Gal）活性的上升,草莓果实软化关键基因FaPG1和FaβGal4在处理后均下调表达。在接种AMF条件下,随着糖醇螯合钙浓度的增加,果实硬度逐渐增强,其中以接种AMF联合施用0.20%糖醇螯合钙溶液效果最好。     

Cloning,Expression and Bioinformatics Analysis of Enterococcus faecalis from Northeast Black Bear

In order to detect Enterococcus faecalis endocarditis antigen (EfaA) of bear that was used to disclose the infected Northeast Black bear immediately, a pair of PCR primers was synthesized by EfaA gene of Enterococcus faecalis that was recorded in GenBank (accession number:U03756.1) and the target fragment was 689 bp. The PCR product was cloned by pMD19-T vector, then was transferred to Escherichia coli DH5α competent cells and the positive clones were filtered. Recombinant plasmid (pMD19-T-EfaA) was extracted. Identification of PCR and digestion, sequencing, the structure prediction of proteins were operated. The results showed that EfaA gene was cloned successfully, and the gene homology with ATCC 29212 was 100.0%. pMD19-T-EfaA was constructed successfully, structure of proteins was predicted. This study provided theoretical basis for diagnostic methods of Enterococcus faecalis and laid basis for prevention and control of Northeast Black bear disease.     

猪乙脑病毒HW株的全基因组克隆及分析

流行性乙型脑炎(Japanese encephalitis virus,JEV)是一种中枢系统人畜共患急性传染病,由蚊为媒介传播,以高热和狂暴或沉郁等神经症状为特征[1],猪患乙型脑炎的主要症状表现为怀孕母猪流产,产死胎或弱仔,公猪则患睾丸炎,仔猪呈神经症状,使养猪业遭受巨大损失。1988年我国学者方     

Embryo-mediated genome editing for accelerated genetic improvement of livestock

Selecting beneficial DNA variants is the main goal of animal breeding. However, this process is inherently inefficient because each animal only carries a fraction of all desirable variants. Genome editing technology with its ability to directly introduce beneficial sequence variants offers new opportunities to modernize animal breeding by overcoming this biological limitation and accelerating genetic gains. To realize rapid genetic gain, precise edits need to be introduced into genomically-selected embryos, which minimizes the genetic lag. However, embryo-mediated precision editing by homology-directed repair (HDR) mechanisms is currently an inefficient process that often produces mosaic embryos and greatly limits the numbers of available edited embryos. This review provides a summary of genome editing in bovine embryos and proposes an embryo-mediated accelerated breeding scheme that overcomes the present efficiency limitations of HDR editing in bovine embryos. It integrates embryo-based genomic selection with precise multi-editing and uses embryonic cloning with elite edited blastomeres or embryonic pluripotent stem cells to resolve mosaicism, enable multiplex editing and multiply rare elite genotypes. Such a breeding strategy would enable a more targeted, accelerated approach for livestock improvement that allows stacking of beneficial variants, even including novel traits from outside the breeding population, in the most recent elite genetic background, essentially within a single generation.     

胡椒4-香豆酸：辅酶A连接酶Pn4CL基因的克隆及表达分析

根据胡椒4-香豆酸：辅酶A连接酶（4-coumarate:coenzyme A ligase, 4CL）基因的部分序列设计引物,运用RACE方法获得其家族成员的1个全长cDNA,命名为Pn4cl,长度2130 bp,开放阅读框1638 bp,编码545个氨基酸。预测Pn4CL分子量为59.57 kDa,理论等电点为5.70。该基因含有AMP-binding（AMP-binding enzyme）、CaiC[Acyl-CoA synthetase (AMP-forming) /AMP-acid ligaseⅡ]、PLN02246、AFD-class I等结合域,具有植物4CL所共有的保守结构域。系统进化分析表明,Pn4CL与北细辛的同源性最高,同时与木兰分支类植物的4CL聚类在一起,与菊分支的进化距离较近,与蔷薇分支的进化距离较远。亚细胞定位表明,该蛋白定位在细胞膜上。Real-time RT-PCR结果表明,该基因受外援激素SA和MeJA诱导表达,同时接种辣椒疫霉菌后,Pn4CL基因的表达量在抗/感2种胡椒中均出现先增加后减少的现象,并且在抗病种质中表达量较高。研究结果为Pn4CL的功能研究提供了理论依据。     

芒果中性/碱性转化酶MiNI基因的克隆与表达分析

采用RT-PCR技术克隆了1个编码NI基因的cDNA序列,命名为MiNI基因,并对不同来源的中性/碱性转化酶的分子特征及系统进化进行比较分析。结果表明：MiNI基因开放阅读框为2034 bp,编码677个氨基酸,相对分子量为76.6 kDa,理论等电点为6.24;生物信息学分析结果显示,NI二级结构α螺旋占38.85%,无规则卷曲占35.45%,伸展链占18.91%,β折叠占6.79%;MiNI具有glycoside hydrolase family 100结构保守域,与克里曼丁桔、龙眼、巴西橡胶树和番木瓜都具有一致的motif位点;MiNI基因编码的氨基酸序列与克里曼丁桔、龙眼氨基酸序列同源性最高;构建NI系统进化树分析表明,与芒果遗传距离最近的是克里曼丁桔,最远的是玉米和枸杞。qRT-PCR分析显示,果皮MiNI基因表达量远高于果肉;花后10~40 d,果皮MiNI基因表达量显著下降;花后40~100 d,果皮MiNI基因表达量维持在一个相对稳定水平;花后100~130 d,随着果实成熟,果皮MiNI基因表达量又显著上升;而花后10~40 d,果肉MiNI基因表达量显著下降,至果实发育后期果肉MiNI基因表达量始终处于极低水平。该研究为进一步了解MiNI基因在芒果果实蔗糖代谢过程中的作用以及从分子角度阐明芒果糖代谢机理奠定理论和技术基础。     

小麦耐冷相关基因TaCTR的克隆及其表达特性分析

低温冷害是小麦生长发育过程中面临的重要非生物逆境因素。为了挖掘小麦耐冷功能基因,本研究采用同源克隆的方法从普通小麦品种小偃22中分离到一个耐冷相关基因TaCTR,该基因序列全长2 192 bp,含有12个外显子、11个内含子,编码区全长为1 407 bp,编码468个氨基酸,分子量约为53.43 kDa;系统进化树分析表明,该基因在进化关系上与山羊草最近;亚细胞定位结果显示,该基因编码的蛋白为膜蛋白;实时荧光定量PCR（RT-PCR）结果表明,TaCTR在不同品种、不同组织和不同发育阶段低温特异表达;在干旱、高盐及GA处理下,TaCTR的表达量显著上升,说明该基因可能参与调控小麦的抗逆反应。     

玉米轴色突变体698-3R的遗传鉴定

以辐射诱变获得的玉米红轴突变体698-3R为材料,通过遗传交配设计分析轴色的遗传规律,利用SSR-BSA法初步定位轴色基因,并对候选基因进行克隆和表达分析。结果表明:(1)突变体698-3R的红轴对白轴性状表现为显性遗传,受一对核基因控制,初步定位在第一染色体上的SSR标记phi095与umc1452之间,与phi095相距3.9cM,与umc1452相距7.1cM,将该基因暂定名为C(t);(2)以轴色基因 P1的cDNA为模板克隆C(t),发现698-3R有3个C(t)转录本,而野生型698-3中至少有2个;预测氨基酸序列比对发现,698-3R中3个C(t)蛋白的氨基酸序列与已知P_1-wr蛋白一致,突变使其获得完整的MYB结构域和酸性激活域,而野生型698-3中698-3-P-1蛋白由于编码序列缺失导致移码突变,不具有该功能结构域;(3)对C(t)基因qRT-PCR分析发现,除25DAP外,其余4个时期在698-3R中表达水平均显著或极显著高于698-3;以 A1和 C2基因表达验证分析发现,C(t)与验证基因表达模式一致,说明C(t)可能具有 P1激活调控 A1和 C2基因表达的功能。推测该红轴基因C(t)可能为一个 P_1-wr基因。