Genome editing is essential for studying gene function for pathogens such as Erwinia amylovora, which causes devastating fire blight in fruit trees of the family Rosaceae. CRISPR/Cas genome editing technology has been widely used in various model organisms, but E. amylovora lacks effective genome editing tools. Here, we present a simple and effective gene knockout method based on the E. amylovora endogenous type I-E CRISPR/Cas system that uses plasmid to express crRNA. The plasmid contains the CRR1 array expression cassette of E. amylovora, the replication initiation region of the E. amylovora plasmid pEA60, and backbone of the cloning vector pMD19. Using this method, we successfully obtained the pEA29 plasmid curing and ams operon deletion mutants. Five distinct deletions ( 33 k to 52 k bp) were identified from 11 plasmid-transformed clones by whole-genome sequencing, with no off-target effects detected. The ams operon knockout mutant, as expected, did not produce amylovoran, the major extracellular polysaccharide component and pathogenic factor of E. amylovora, and was non-pathogenic in plants. Thus, this method allows for quick and convenient construction of genetic mutants of E. amylovora using plasmid-expressed crRNAs.

Southern tomato virus (STV) is a dsRNA virus, which belongs to the newly formed Amalgavirus genus of the Amalgaviridae family. Currently there is no report regarding the presence of STV in tomato tissues. In this study, we performed in situ hybridization to examine the distribution of STV in host tissues. STV was found in the leaves, stems, seeds, shoot apexes and root tips of tomato and localized in the cortex tissue, vascular tissues, pith, seed coat, endosperm, cotyledon (including inner cotyledons and outer cotyledons), hypocotyls and radicles of infected tomato tissues. In addition, STV was detected in the apical part of the stems and roots for the first time. This indicates that STV is a systemic infectious virus.

【Objective】The purpose of this study was to establish a simple and efficient approach for identifying all T-DNA insertion sites.【Method】A T-DNA insertion sites analysis approach based on high-throughput sequence technologies was developed, called PSORA: Parallel sequencing of one round amplicons. The process involves high-throughput amplicon sequencing of a round of thermal asymmetric PCR (TAIL-PCR) and bioinformatics analysis of T-DNA insertion sites, which reduces concerns about the specificity of TAIL-PCR. In PSORA, only two primers are required, a degenerate primer and a T-DNA specific primer. A 6-nt Barcode was designed at the 5’ end of the specific primers for labeling different transgenic events. All five transgenic events (L1, L6, L9, L15 and L19) of tobacco used in this study were produced via Agrobacterium mediated transformation with plasmids pBI121. In addition, the results of PSORA are confirmed by standard PCR.【Result】The T-DNA insertion sites of five transgenic events were analyzed by PSORA. The results showed that L6 contained two insertion sites (36 316 bp on NW_015801367 and 42 202 bp on NW_015950898), the lines of L9, L15 and L19 each contained one insertion site (The insertion site of L9 was located at 235 969 bp on NW_015943682. The insertion site of L15 was located at 60 529 bp on NW_015802951 and the insertion site of L19 was located at 12 188 bp on NW_015863435), but the insertion site of L1 could not be detected. PCR was performed to validate the results from bioinformatics analysis, transgenic events with different insertion sites were used as negative controls for each other, and the wild type (WT) was used as a blank control. The results showed that specific amplification consistent with expectations was obtained in each transgenic event. The effectiveness of PSORA was successfully confirmed.【Conclusion】PSORA is an effective strategy to analyze T-DNA insertion sites. PSORA can parse the comprehensive molecular characteristics of all T-DNA insertion events simultaneously, making it simpler and faster than the traditional methods of genome walking.

《分子生物学》是生命科学的前沿，是生物类专业高等院校本科生的专业基础课。根据新时代高等教育的使命，从《分子生物学》课程的教学内容、教学方法和课程考核等多个方面重构了课程的教学体系，有利于提高课程的教学质量和教师的教学水平，为培养复合型专业人才奠定了良好的基础。

Abstract Southern tomato virus (STV) is a dsRNA virus, which belongs to the newly formed Amalgavirus genus of the Amalgaviridae family. Up to now, there is no report on the presence of STV in tomato tissues. In this study, we performed in situ hybridization to examine the distribution of STV in host tissues. The STV was found in the leaves, stems, seeds, shoot apexes and root tips of tomato and localized in the cortex tissue, vascular tissues, pith, seed coat, endosperm, cotyledon (including inner cotyledons and outer cotyledons), hypocotyls and radicles of infected tomato tissues. In addition, STV was detected in the apical part of the stems and roots for the first time. This indicates that STV is a systemic infection disease for tomato.

思政教育是立德树人的灵魂工程.以细胞生物学专业知识与社会主义核心价值观为主要育人内容,能够深化课程内涵,提高课程教学质量,赋予课程思政活力.探索在教学的各个环节实施课程思政的途径和方法,把立德树人贯穿始终,旨在引导学生树立正确的国家观、民族观,为中国特色社会主义事业培养合格建设者和可靠接班人.

采用高通量测序(High-throughput sequencing,HTS)技术检测新疆蟠桃感染病毒的情况.采集具有穿孔、脉间褪绿等症状的蟠桃嫩叶,提取总RNA,用于高通量测序,共获得13.36 Gb的数据,包含44 563 187对reads,其中比对到油桃茎痘相关病毒(nectarine stem pitting-associated virus,NSPaV,KT273409)和亚洲李属病毒(asian prnnus virus,APV2,KT893294)基因组的分别有9 943对和40 036对,经拼接各得到长4 978和9 393 nt的contig,基因组覆盖率均接近100％(5'端分别差10个和7个碱基),核酸一致度分别为95.0％和92.9％,将此分离物分别命名为NSPaV-Tao和APV2-Tao4.用RT-PCR方法对23个蟠桃树样品进行了检测,结果NSPaV和APV2检出率分别为43.5％和69.6％.通过比较己知NSPaV病毒的核苷酸序列,发现NSPaV-Tao可能是其他分离物重组的结果.

为明确新疆石河子辣椒感染病毒的种类,对表现明显皱缩、褪绿和卷叶等症状的辣椒样本进行长链非编码RNA(Long noncoding RNA,LncRNA)测序.根据LncRNA测序和RT-PCR检测,发现甜椒内源RNA病毒(Bell pepper endomavirus,BPEV)、辣椒隐症病毒2(Pepper cryptic virus 2,PCV2)和辣椒轻斑驳病毒(Pepper mild mottle virus,PMMoV)3种病毒.RT-PCR检测数据显示,新疆石河子的辣椒普遍感染BPEV和PMMoV.将BPEV的RdRp基因序列和PCV2-RNA1包含RdRp基因的序列分别进行核苷酸序列相似性和系统进化分析.结果 表明本研究中BPEV-XJ和PCV2-XJ与其它分离物的相似性分别为89.1％ ～97.3％和96.9％～ 99.7％.BPEV-XJ与BPEV-TW分离物亲缘关系最近;PCV2-XJ与PCV2-DR分离物亲缘关系最近.辣椒中检测到BPEV和PCV2为新疆首次报道.

3型视神经萎缩蛋白(Optic atrophy type 3,OPA3)普遍存在于真菌、植物和动物中,但它在植物中的功能尚不清楚.本研究构建了番茄OPA3-like(SlOPA3L)基因的过表达载体35S::SlOPA3L并遗传转化烟草.与野生型烟草叶片相比,过量表达SlOPA3L基因的转基因烟草的子叶呈花叶状、部分组织失绿,转基因烟草的少数早期真叶呈花叶表型,随着叶片发育后又逐渐恢复正常.转基因烟草的叶片细胞表型分析表明:真叶花叶的叶片的海绵组织和栅栏组织发育受到显著的影响.这些结果表明:SlOPA3L基因可能影植物叶片细胞的分裂和参与植物叶片发育早期的叶绿体形成.

为培育成熟果实软化程度低,货架期长的番茄植株,以加工番茄甘露糖苷酶基因（α-Man）为编辑对象,设计由番茄U6启动子驱动、长21 bp的guide RNA（gRNA）指导hCas9核酸酶,靶向编辑α-Man的第1个外显子。首先构建基于CRISPR/Cas9系统的植物表达载体,并通过农杆菌介导的遗传转化获得加工番茄转基因株系,然后取转基因番茄叶片基因组DNA,利用限制性内切酶法结合PCR扩增对α-Man编辑位点附近的DNA片段进行检测及测序分析。结果表明,14株转基因番茄植株有2株检测到突变现象。α-Man突变体TA克隆测序结果显示有2种编辑类型,一种表现为52 bp的缺失突变;另一种表现为单碱基突变。实现了对番茄α-Man的编辑。

RNA sequencing (RNA-Seq)-based gene expression analysis is applicable to a wide range of biological purposes in various species. Reverse transcription quantitative PCR (RT-qPCR) is also used to assess target gene expression utilizing stably expressed reference genes as internal control under a given set of conditions. However, investigations of the reference genes for RT-qPCR normalization in the process of somatic embryogenesis (SE) initial dedifferentiation in Gossypium hirsutum are rarely reported. In this study, on the basis of our previous transcriptome data of three different induction stages during SE initial dedifferentiation process in four G. hirsutum cultivars that have different SE capability, 15 candidate genes were selected during SE initial dedifferentiation process, and their expression stability was evaluated by geNorm, NormFinder, and BestKeeper. The results indicated that the two genes of endonuclease 4 (ENDO4) and 18S ribosomal RNA (18S rRNA) showed stable expression in the four different G. hirsutum cultivars, endowing them to be appropriate reference genes during three induction stages in the four cotton cultivars. In addition, the stability and reliability of the two reference genes of ENDO4 and 18S rRNA were further verified by comparing the expressions of auxin-responsive protein 22 (AUX22) and ethylene-responsive transcription factor 17 (ERF17) between RT-qPCR results and the RNA-seq data, which showed strong positive correlation coefficient (R2 = 0.8396–0.9984), validating again the steady expression of ENDO4 and 18S rRNA as the reliable reference genes. Our results provide effective reference genes for RT-qPCR normalization during SE process in different G. hirsutum cultivars.

为培育成熟果实软化程度低,货架期长的番茄植株,以加工番茄甘露糖苷酶基因(α-Man)为编辑对象,设计由番茄U6启动子驱动、长21 bp的guide RNA(gRNA)指导hCas9核酸酶,靶向编辑 α-Man的第1个外显子.首先构建基于CRISPR/Cas9系统的植物表达载体,并通过农杆菌介导的遗传转化获得加工番茄转基因株系,然后取转基因番茄叶片基因组DNA,利用限制性内切酶法结合PCR扩增对 α-Man编辑位点附近的DNA片段进行检测及测序分析.结果表明,14株转基因番茄植株有2株检测到突变现象.α-Man突变体TA克隆测序结果显示有2种编辑类型,一种表现为52 bp的缺失突变;另一种表现为单碱基突变.实现了对番茄 α-Man的编辑.

Analysis of gene expression level by RNA sequencing (RNA-seq ) has a wide range of biological purposes in various species. Real-time fluorescent quantitative PCR (qRT-PCR) evaluated gene expression levels and validated transcriptomic, which will depend on the stably expressed reference genes for normalization of the gene expression level under specific situations. In this study, 15 candidate genes were selected from transcriptome datasets during somatic embryogenesis (SE) initial dedifferentiation in Gossypium hirsutum L. of different SE capability. To evaluate the stability of those genes, geNorm, NormFinder and BestKeeper were used. The results revealed that ENDO4 and 18srRNA could be as appropriate reference genes under all conditions. The stability and reliability of the reference genes were further tested through comparison of qRT-PCR results and RNA-seq data, as well as evaluation of the expression profiles of auxin-responsive protein (AUX22) and ethylene-responsive transcription factor (ERF17). In summary, the results of our study indicate the most suitable reference genes for qRT-PCR during three induction stages in four cotton species.

[目的]验证基于CRISPR/Cas9系统构建的靶向编辑加工番茄(Solanum lycopersicum)elF4E1基因载体的有效性,为CRISPR/Cas9系统在培育PVY抗性植株中的应用提供技术支持.[方法]构建靶向编辑番茄真核翻译起始因子elF4E1基因的CRISPR/Cas9系统表达载体,用农杆菌渗透法瞬时转化番茄植株,PCR扩增已转化植株靶位点周围DNA序列后用HaeⅢ进行酶切,回收未切开的条带与pGEM-T载体连接后进行单克隆测序.[结果]对测得的9个克隆序列进行比对分析,在PAM(protospacer adjacent motifs)上游的6～8 bp的碱基处均发生突变,并且都为单碱基的替换,导致多肽链中单个氨基酸的替换.[结论]利用CRISPR/Cas9基因组编辑系统构建的载体能够特异性地靶向加工番茄eIF4E1基因,为利用CRISPR/Cas9系统敲除eIF4E1基因,获得抗PVY病毒的番茄育种材料奠定了基础.

在进行酵母双杂交试验时发现,马铃薯Y病毒HC-Pro蛋白与番茄未知蛋白发生相互作用,经测序、Blast比对,确认其为番茄叶绿素a/b结合蛋白Cab-1A,Genebank序列号为XM_010318610.1.为初步验证这一发现,设计引物,然后以番茄cDNA文库为模板,克隆出798 bp的cab-1a全长序列,并构建了cab-1a基因的亚细胞定位载体pSPGFP-Cab-1A,并运用叶盘法将其转化到烟草表皮细胞中,在荧光共聚焦显微镜下观察其定位情况.结果表明,cab-1a基因编码的Cab-1A-like蛋白位于细胞核和细胞质中,为与马铃薯Y病毒(potato virus Y,PVY)蛋白HC-Pro(helper component proteinase)相互作用的后续研究奠定理论基础.

为了获得来源于西洋梨红贝雷沙寄主潜带苹果茎沟病毒(ASGV)分离物的基因组全长序列,并明确其分子特性,采用RT-PCR、RACE末端克隆及生物信息学方法,对ASGV分离物基因组全长进行序列测定,并对其序列特点进行分析.结果发现,来源于西洋梨的ASGV-HB分离物的基因组全长序列为6496 nt(GenBank登录号:KU605672),含有2个开放阅读框ORF1、ORF2及2个可变区Ⅵ、Ⅶ.序列分析表明ASGV-HB与GenBank登录的18个ASGV分离物的基因组全长核苷酸序列同源性为80.6%~87.7%;ORF1和ORF2编码的氨基酸同源性分别为84.3%~92.3%和94.4%~98.7%;Ⅵ和Ⅶ可变区的氨基酸序列同源性分别为22.9%~68.8%和48.8%~92.2%;系统发育树分析显示,来源于不同国家的相同寄主的ASGV分离物聚集为同一分支.结果表明,ASGV的分子变异无地域相关性,具有一定的寄主选择性,研究结果为进一步探究ASGV的群体遗传进化机制及其防治提供了重要的分子信息.

[目的]获得高效表达的类视神经萎缩蛋白3(OPA3-like),并分析其生物信息学功能.[方法]利用Vector NTI和ExPASy,SignalP,Plant-mPLoc等在线工具对OPA3-like蛋白基本理化性质、信号肽、跨膜区及亚细胞定位进行分析;构建原核表达载体pET22b-OPA3-like、pET28a-OPA3-like、pET32a-OPA3-like并分别转化至大肠杆菌BL21(DE3)和Rosetta(DE3)中,经IPTG诱导融合蛋白表达,SDS-PAGE对结果进行验证.[结果]OPA3-like蛋白是不稳定蛋白,含有跨膜区,可能位于叶绿体;原核表达表明,对于同一重组质粒而言,融合蛋白在两种菌中的表达量无明显差别,其中,转化pET28a-OPA3-like可表达20kDa的融合蛋白;转化pET32a-OPA3-like可表达37kDa的融合蛋白,且主要都以包涵体形式存在,转化pET22b-OPA3-like无融合蛋白的表达.[结论]转化pET32a-OPA3-like的表达菌可以高效表达融合蛋白(37 kDa),有利于进一步研究OPA3-like在植物中的亚细胞定位及功能.

细胞工程是生物工程的一个重要方面,也是发展迅猛的前沿学科之一.为了增强学生的实践创新能力及动手能力,配合细胞工程理论课教学改革的不断深化,细胞工程实验教学改革已是大势所趋.本教研组经过多年的探索,从教学内容、教学条件和考核制度等方面,对细胞工程实验教学进行了改革,旨在提高学生的综合实验能力,培养他们独立思考、科研创新的能力.