Genetics is the subject of studying heredity and variation,which has strong practicality and is closely related to human life.In order to mobilize students′ interest in learning,enhance their understanding and memory of knowledge points,inspire their thinking ability,and then improve the teaching quality,adopted the five-step teaching method（designing the situation and set questions,classroom teaching and show results,key teaching,give directions and extension,consolidation exercise,inspection and evaluation,induction and summary,strengthen memory）to organize teaching activities.Expounds the application of five-step teaching method in the course of genetics,providing reference for student training in basic biology sciences.

基于人参、三七基因组测序数据,在基因组水平上对SE基因家族进行鉴定,对其系统进化关系、基因结构、顺式作用元件以及SE基因复制事件等进行分析.结果表明,人参、三七的基因组中共鉴定到20个SE基因,聚为5个分支.SE基因在人参和三七中高度保守,所有SE均包含SE域和FAD/NAD(P)结合域.人参、三七的SE均含有干旱诱导元件、低温响应元件、茉莉酸甲酯响应元件、脱落酸响应元件等参与对植物激素和非生物胁迫响应的关键顺式调控元件.适应性进化分析结果显示,人参和三七SE基因在自然选择中是以纯化选择为主,人参和三七SE 基因在进化过程中高度保守.

目的:研究冠心病患者的指纹纹型分布,并与正常人群指纹分布作比较,为临床医学提供基础皮纹学参数.方法:对160例唐山地区冠心病患者及160例对照组人群印泥拓取法采集指纹,放大镜下鉴定.结果:冠心病患者的指纹参数值为:斗形纹(W)占56.19％,箕形纹(L)占41.25％,弓形纹(A)占2.56％.A形纹多见于示指,其次是中指;Lr多见于示指;Lu多见于小指;W多见于环指.冠心病患者示指箕形纹和弓形纹的频率较对照组具有显著性差异.双手十指同纹型的频率为20％.一手五指纹型组合频率为OLW(65.31％)>同型组合(26.25％)>ALO(4.69％)>ALW(3.75％).对应手指纹型组合频率为W/W(49.13％)>L/L(33.00％)>L/W(13.00％)>A/L(3.50％)>A/A(1.25％).患者组和对照组示指出现A/L和L/W组合的频率差异显著,2组中L/W和W/W组合的频率差异显著.结论:冠心病患者指纹纹型分布具有特异性,且男性和女性间差别不显著.

传统的生物技术专业实践教学已难以适应新时代生物技术产业的需求。文章基于OBE理念，重构了生物技术专业本科人才培养的实践课程教学体系和教学设计，多措并举地开展了“课内”和“课外”相结合的多种形式的实践教学，形成了科学的实践教学评价、考核方式。为培养成满足产业需求的高素质应用型生物技术人才奠定了基础。

目的 研究根皮苷对食管癌细胞系表达谱的影响,探索根皮苷作用于食管癌细胞系的信号通路及其潜在功能.方法使用根皮苷处理人食管癌KYSE450细胞,提取总RNA并建立测序文库,进行转录组测序;使用DESeq2程序鉴定差异表达基因,对差异表达基因进行基因本体(gene ontology,GO)功能及京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析;使用MCODE进行差异基因核心模块筛选,使用GEPIA数据库分析核心模块中的基因对食管癌生存的影响;使用TIMER在线分析根皮苷对食管癌组织免疫细胞浸润的影响.结果 转录组测序结果显示,根皮苷处理后的食管癌细胞具有4602个差异表达基因,其中2407个为上调基因,2195个为下调基因.通路富集分析显示,根皮苷对蛋白质加工、胰岛素抵抗、基因复制和细胞周期等信号通路产生影响.在差异表达基因核心模块中,E3泛素蛋白连接酶2(E3 ubiquitin ligase mind bomb 2,MIB2)高表达可降低食管癌患者的总体生存率;环指蛋白19B(ring finger protein 19B,RNF19B)、三重基序蛋白69(tripartite motif-containing protein 69,TRIM69)、泛素连接酶(ubiquitin conjugating enzyme,UBC)和克隆 E3泛素连接酶(homologous to the E6-associated protein carboxyl terminus domain containing 2,HECTD2)的表达水平可影响食管癌癌组织的纯度以及免疫细胞浸润的类型.结论 根皮苷可影响食管癌细胞的转录谱,其差异表达基因主要集中在细胞生长相关的信号通路.

目的 筛选和鉴定刺五加Eleutherococcus senticosus中的细胞色素P450基因(EsP450),分析其进化特征,探究其与刺五加总皂苷含量的相关性.方法 根据转录组测序结果,筛选得到EsP450基因,并对其进行生物信息学分析与适应性进化分析,采用qRT-PCR法检测EsP450基因的表达量,采用分光光度计测定刺五加的总皂苷含量.结果 筛选得到了18条EsP450基因.刺五加P450蛋白不存在跨膜区域,二级结构以α-螺旋和无规则卷曲结构为主.EsP450基因不存在正选择位点.各EsP450间的表达量差异显著,部分基因表达量差值在10倍以上,7条EsP450基因的表达与皂苷含量呈正相关关系(P＜0.05).结论 鉴定出与刺五加总皂苷含量存在正相关关系的EsP450基因,EsP450基因在进化中受到纯净选择.

目的 克隆多穗柯Lithocarpus polystachyus的无色花青素还原酶(leucoanthocyanidin reductase,LAR)基因,并对其表达量与根皮苷含量的关系进行分析.方法 根据多穗柯转录组测序的结果(Unigene号:DN3071 1_c0_g1_j1),利用PCR法扩增获得多穗柯LAR基因的全长cDNA序列,进行生物信息学分析,采用实时荧光定量PCR (qRT-PCR)法检测LAR基因的表达量,使用UPLC法测定根皮苷的含量,应用SPSS 18.0软件分析LAR基因表达量与根皮苷含量间的相关关系.结果 多穗柯LAR基因的cDNA全长1053bp,包含一个完整的开放阅读框,编码350个氨基酸.该蛋白不存在跨膜区域,定位于细胞质中.多穗柯LAR蛋白属于PCBER_SDR_a家族,与栓皮栎LAR蛋白具有较高的相似性(95％),且亲缘关系最近.多穗柯的根皮苷含量与LAR基因的表达量呈正相关(P＜0.05)关系.结论 首次克隆得到多穗柯LAR基因,并证实多穗柯根皮苷含量与LAR基因的表达量呈正相关,为揭示多穗柯中根皮苷的生物合成机制奠定理论和技术基础.

目的 克隆刺五加香叶基焦磷酸合成酶(geranyl pyrophosphate synthase,GPS)基因的cDNA序列并分析基因序列特征、不同器官中基因表达水平及其与皂苷含量的相关性.方法 提取刺五加的RNA,逆转录为cDNA.根据转录组测序结果中编码GPS的unigene(c37362.graph_c0),设计特异性引物,经过PCR扩增GPS基因的cDNA全长.利用实时荧光定量PCR (qRT-PCR)分析GPS基因在不同器官中的表达水平,并通过分光光度法检测刺五加总皂苷含量.结果 克隆得到长1 260 bp、编码419个氨基酸的刺五加GPS基因cDNA.GPS蛋白定位于线粒体内且不存在跨膜区域.GPS基因在各个器官中均有表达,在叶片中的表达量最高,是根中表达量的5.26倍.GPS基因的相对表达量与皂苷量呈现出同升同降的变化趋势,表现为显著正相关关系(r=0.851,P＜ 0.05).结论 首次克隆获得刺五加GPS基因的cDNA全长序列,明确了刺五加GPS基因的表达量与皂苷含量之间存在正相关关系.

通过对2016级生物技术、生物信息专业90名学生遗传学多元化考核评价体系的实施,改变以往的教学模式和考核方式,探究课堂教学中存在的问题和不足并采取相应的措施,以期全面评价学生的综合能力.该考核评价体系包括平时作业、翻转课堂和易位式教学、阶段性测验3个考核项目,并对每个考核项目进行了细化和量化,具有一定的可行性.

为了克隆多穗柯4-香豆酸辅酶A连接酶(4-coumarate-CoA ligase,4CL)基因,了解其表达情况,从而为深入研究4CL基因分子功能及黄酮类化合物生物合成奠定基础,依据多穗柯转录组测序结果,设计特异性引物,PCR扩增得到4CL基因cDNA全长序列,利用相关软件进行生物信息学分析;采用qRT-PCR法检测4CL基因在不同器官中的表达情况,使用SPSS18.0软件分析其表达量与根皮苷含量间的关系.结果表明:多穗柯4CL基因cDNA全长1704 bp,包含长1629 bp的开放阅读框,编码含542个氨基酸的蛋白质.该蛋白定位于细胞质中,属亲水性蛋白.多穗柯4CL基因在除根以外的各个器官中均有表达,且表达量与根皮苷含量间呈极显著的正相关关系(P<0.01).

目的 对五加科药用植物的鲨烯合酶(squalene synthase,SS)进行适应性进化分析.方法 利用PAML软件的分支模型、位点模型、分支-位点模型以及MEC模型、SLAC、FEL和REL等方法对五加科7种药用植物的23条SS基因进行适应性进化分析.结果 在PAML与MEC模型的分析中发现多数分支与位点处于较强的负选择下,未发现正选择位点;通过SLAC、FEL和REL分析同样显示存在大量的负选择位点,仅412P、413N、415K为正选择位点.结论 负选择对五加科SS基因起主导作用,而发现的412P、413N、415K位点可能与SS的活性有关.

采用平板纸片法测定药用植物刺五加内生放线菌菌株13-12发酵液抑菌活性及稳定性;PCR扩增、克隆并测序分析菌株活性物质合成相关功能基因;并通过生理生化、培养特征及16S rRNA进行分类鉴定.结果表明:菌株13-12发酵液对金黄色葡萄球菌、枯草芽孢杆菌、草分枝杆菌、MRSA均有不同程度的抑制作用,在4～20℃、pH6.0～8.0及光照48 h条件下发酵液抑菌活性稳定;菌株基因组含有NRPS、Halo基因;通过菌株表型特征及分子特征初步鉴定该放线菌为游动单胞菌属(P lanomonos pora)有效发表种球形浮游动单胞菌(Planomonospora sphaerica)的一个菌株.菌株有合成β-内酰胺类抗生素等多种活性物质的潜能,具有进一步开发利用价值.

利用生物信息学方法比较壳斗科6个属14个物种的叶绿体基因组间差异,以近缘物种榛为外类群构建系统进化树,揭示壳斗科叶绿体基因组的结构特征及变异规律。结果显示,14种壳斗科植物的叶绿体基因组均为双链环状分子结构,大小在160 kB左右,差异较小,最大仅差1 366 bp;基因顺序基本一致,而基因数量有所差异,infA、petG、rpl22、ycf1、ycf15等多个基因在部分物种中发生丢失;主要有32个蛋白编码基因长度发生变异,其原因是内含子的丢失、内含子或者编码区的长度改变,华南锥基因长度变异较大; 4个IR边界相对保守,但锥栗、Castanea pumila、华南锥3个物种由于边界扩张导致rps19基因部分序列进入到IR区;以榛为外类群构建的系统发育树,各进化支支持率较高,分辨率较好。研究结果表明,叶绿体基因组可以用于分析关系较近与进化较快物种的系统发生问题,为系统发育和进化研究提供依据。

目的 研究刺五加内生放线菌株13-85抑菌活性及活性稳定性,检测菌株活性物质合成相关功能基因,对菌株进行鉴定.方法 采用平板纸片法测定菌株抑菌活性及发酵液稳定性;PCR扩增其活性物质合成相关功能基因片段(PKSⅠ、PKSⅡ、NRPS、Halo、CYP),克隆并测序分析.通过菌株表型特征及分子特征对其鉴定.结果 菌株13-85发酵液具有广谱较强的抗病原菌活性,抑菌活性较为稳定;菌株基因组含有PKS Ⅱ、NRPS基因.经比对,PKS Ⅱ片段与已知Streptomyces fradiae PKS序列(登录号AFO70128.1)相似度为89％,NRPS片段与Streptomyces virginiae NRPS序列(登录号WP_033225509.1)相似度达95％.菌株13-85与Streptomyces amritsarensis(登录号GQ906975)16 S rRNA基因序列相似率高达99.65％.结论 菌株13-85发酵液有较为广泛的抑菌活性且稳定性较好,菌株含有PKSⅡ、NRPS基因,初步鉴定该放线菌为链霉菌属有效发表种Streptomyces amritsarensis的一个菌株,具进一步开发应用价值.

Objective To understand the chalcone isomerase (CHI) gene expression by cloning it in Lithocarpus polystachyus.Methods A full-length cDNA of CHI gene from Lithocarpus polystachyus (Lpr-CHI) was obtained by PCR cloning technique according transcriptomics sequences infromation,which bioinformatics analysis was carried out.The expression of CHI gene in different organs ofLithocarpus polystachyus was detected by qRT-PCR.Results Lpr-CHI was 772 bp in full length with an open reading frame (ORF) of 696 bp,which encoded a protein with 231 amino acids.The protein did not contain a transmembrane domain and is localized in the cytoplasm.Lpr-CHI gene expression was found in different parts,and reached the highest in leaf,which was 9.75 times of the least gene expression in root.Conclusion Lpr-CHIwas obtained for the first time,and it was clear that the gene belongs to CHItype Ⅱ.And the expression of Lpr-CHI in each organ was significantly different.

Objective To obtain the distribution and functional activity of CpG islands in promoters ofFPS,SS,and SE from Eleutherococcus Senticosus.Methods Based on the promoter sequence ofFPS,SS and SE,CpG islands were predicted by using EMBOSS and Li Lab.The functional verification of transformed Arabidopsis thaliana was mediated by Agrobacterium tumefaciens by using GUS and pC AMBIA 1301 plasmid as the reporter gene and expression vector respectively.Results Two CpG islands were found in FPS and SS promoter with the lengths of 520 bp,218 bp and 108 bp,103 bp,and three CpG islands in SE promoter in 290 bp,119 bp and 149bp.The promoters of FPS,SS and SE all had promoter activities at different level,in which SS promoter was the highest one.Conclusion The functional verification and distributions of CpG islands in the promoters area of FPS,SS,and SE were reported in this research at first time,which established the foundation for the methylation analysis ofFPS,SS,and SE and the further studying of the mechanism expression regulation in E.Senticosus.

Objective To clone flavanone 3-hydroxylase (F3H) gene from Lithocarpus polystachyus,and to understand its gene characteristics and initially investigate its expression level in different organs.Methods The total RNA and genomic DNA from blade of L.polystachyus were extracted.Based on the result of RNA-seq,a pair of specific primers were designed.cDNA and DNA sequences of F3H gene from L.polystachyus were amplified by PCR,then bioinformation analysis was performed after sequencing.The expression level of F3H gene in different organs ofL.polystachyus was detected by qRT-PCR.Results The full length of cDNA of F3H gene was 1 340 bp containing a 1 092 bp open reading frame that encoded 393 amino acids,and F3H was located in the cytoplasm.The result of qRT-PCR showed that F3H gene expressed in different organs ofL.polystachyus,and the expression levels of F3H gene were significantly different in different organs (P ＜ 0.05).Conclusion The F3H gene ofL.polystachyus was cloned and its bioinformation was analyzed for the first time,proving that the expression level ofF3H gene in different organs ofL.polystachyus was different.This finding lays a foundation for the studies on secondary metabolism of flavonoids in L.polystachyus.

Be based on the DNA sequence ofsqualene synthase gene 2 (SS2) and squalene epoxidase gene 1 (SE1),using southern blotting and qRT-PCR technique to confirm the copy number of SS2 and SE1 gene.In this research we used the actin gene for reference gene,using qRT-PCR technique and spectrophotometry to confirm gene expression and saponins content of SS2 and SE1 gene in different copy number.The results show that SS2 gene the type of copy number is 1 and 2,and SE1 gene is 1,2,3 and six genotypes by their intercombination.The gene expression and saponins content of SS2 and SE1 gene are improved significantly (P＜0.05) with the improving of copy number in Eleutherococcus senticosus.It has significantly positive correlation (P＜0.01) SS2 and SE1 gene every addional one copy can improve 0.57 and 0.42 times saponins content.Research results established laid the foundation for reveal molecular mechanism that form the difference of saponins content in E.senticosus.

In this paper,based on the cDNA sequence of squalene epoxidase (SE) gene in E.senticosus,we designed specific primers.PCR and TAIL-PCR were used to amplify DNA full-length sequence of SE.Then combined with Plant CARE and other softwares,bioinformatics analysis was performed on the functional elements in the promoter.DNA and promoter sequence of E.senticosus SE gene with 6 307 bp was cloned,containing 8 exons,7 segment introns and untranslated regions at both ends and encoding 554 amino acids.The upstream promoter sequence was 1 907 bp long,containing 36 TATA-box and 27 CAAT-box.Besides,it also contained a variety of cis-acting elements,such as jasmonic acid regulatory element,abscisic acid regulatory element,and light regulatory elements,which indicated that the expression of SE gene was regulated by many factors,such as plant hormone,light and temperature.

以刺五加嫩叶为试材,采用二代测序方法Illumina HiSeq 4000进行转录组测序和生物信息学方法,找出其转录因子,并随机挑选4个不同家族的转录因子,针对6个生长时期和3个器官的样本,使用实时荧光定量PCR确认转录因子的正确性.结果表明:刺五加转录组共计8.34 Gb,拼接得到77 087条Unigenes,有485个转录因子,分类于36个家族.通过qRT-PCR分析,Unigene 49771、Unigene 22314、Unigene 13139和Unigene18837在不同生长发育时期和器官中表达差异极显著(P＜0.01).