Knock-in of exogenous sequences based on CRISPR/Cas9 targeting autosomal genes and sex chromosomes in the diamondback moth,Plutella xylostella

Genetic pest control strategies based on precise sex separation and only releasing sterile males can be accomplished by site-specific genome editing.In the current study,we showed that the mutation of single-allele Pxfl(2)d can significantly impair the normal mating behavior and testis development in male adults of the notorious cruciferous insect pest Plutella xylostella,in addition to its known functions in the ovarian development in female adults and egg hatching.Subsequent CRISPR/Cas9-based knock-in experiments revealed that site-specific integration of an exogenous green fluorescent protein(GFP)gene into autosomal Pxfl(2)d for labelling mutants could be achieved.However,this gene is not a suitable target for GFP insertion to establish a genetically stable knock-in strain because of the severe decline in reproductive capacity.We further screened for the W-chromosome-linked and Z-chromosome-linked regions to test the knock-in efficiency mediated by CRISPR/Cas9.The results verified that both types of chromosomes can be targeted for the site-specific insertion of exogenous sequences.We ultimately obtained a homozygous knock-in strain with the integration of both Cas9 and cyan fluorescent protein(CFP)expression cassettes on a Z-linked region in P.xylostella,which can also be used for early sex detection.By injecting the sgRNA targeting Pxfl(2)d alone into the eggs laid by female adults of the Z-Cas9-CFP strain,the gene editing efficiency reached 29.73%,confirming the success of expressing a functional Cas9 gene.Taken together,we demonstrated the feasibility of the knock-in of an exogenous gene to different genomic regions in P.xylostella,while the establishment of a heritable strain required the positioning of appropriate sites.This study provides an important working basis and technical support for further developing genetic strategies for insect pest control.

实验室果蝇(Drosophil melanogaster)唾腺染色体制备前期技术优化

经过多年的实践积累,从果蝇(Drosophil melanogaster)品系的选择、培养基的配制和果蝇的饲养等方面摸索出了一套改进的制备果蝇唾腺染色体的经验方法。

基于自适应MOEA/D的柔性车间多目标联合优化调度

为了实现柔性作业车间完工时间、机器负荷、交货延期时间、车间能耗等多个目标的联合优化,提出了基于自适应惩罚MOEA/D(multi-objective evolutionary algorithm based on decomposition)的柔性车间多目标调度方法。对具有多个生产机床、多个加工任务、多道工序的柔性车间调度问题进行了描述并建立了优化模型。给出了基于MOEA/D算法的柔性车间调度方法,针对常值惩罚因子无法满足不同邻域对收敛性和染色体多样性不同调整需求的问题,提出了能够随邻域染色体密度自适应调整的惩罚因子,并制定了基于自适应惩罚MOEA/D算法的柔性车间调度流程。在具有8个机床、8个工件共28道工序的生产调度实验中,自适应MOEA/D算法搜索的Pareto前沿解能够支配标准MOEA/D和改进NSGA-Ⅱ算法的Pareto前沿解;在等权重最优解的生产实验中,自适应MOEA/D算法调度方案的完工时间、机器负荷、交货延期时间、车间能耗均小于标准MOEA/D算法和改进NSGA-Ⅱ算法。实验结果证明了自适应惩罚MOEA/D算法在柔性车间调度中的有效性和优越性。

A、D组染色体对普通小麦光合碳同化特性的影响

系统研究了普通小麦（TriticumaestivumL．）中国春A、D组端二体的光合特性。结果表明，4A的双臂对光合速率、光合速率高值持续期、叶绿素含量、叶肉导度均具显著的正效应，1A的短臂和6D的双臂对光合速率和光合速率高值持续期也具正效应，但4D的长臂对光合速率、光合速率高值持续期和RuBPCase活性具负效应。2A的短臂、5A的长臂、6D的长臂和7D的长臂对RuBPCase活性及含量具正效应。因此认为4A、4D等染色体对小麦的光合功能影响较为显著。

小麦(T.aestivum L.)D基因组的研究进展

普通小麦是一个异源六倍体物种,具有ABD三个染色体组,D染色体组在来源和进化过程中都与A、B染色体组不同,D染色体组来自于粗山羊草,含有丰富的抗病、抗虫、抗寒、优质等有益基因,因此D染色体组的研究对小麦的产量、品质改良具有重要意义。但是由于长期的定向遗传改良,我国普通小麦的遗传差异较小,遗传基础狭窄,特别是在D染色体组上尤为突出。一些对作物产量和品质有益的基因未被挖掘利用。本文对小麦D基因组的起源、遗传多样性和拓宽遗传基础的方法及基因定位等进行了综述,并结合本实验室的研究工作对其研究前景进行展望。

小麦生长素结合基因TaABP1-D的表达与染色体定位

生长素影响了植物生长发育的诸多过程。生长素结合蛋白ABP1(auxin binding protein)作为一种生长素受体,在质膜上生长素诱导的快速反应中起重要作用。小麦中已经克隆获得了TaABP1-D,但其在细胞中的作用位置以及在染色体的定位情况仍不明确。本研究利用洋葱表皮细胞瞬时表达系统对小麦生长素结合基因TaABP1-D进行亚细胞定位,结果表明TaABP1-D蛋白为膜蛋白,存在于细胞质和细胞膜中;同时利用中国春缺体-四体材料和信息学方法,将TaABP1-D定位在小麦5D染色体长臂的近着丝粒位置上,距两侧EST标记BE490079和BE405060的遗传距离分别为0.51 c M和0.28 c M。

果蝇内含子和编码序列的信息参数D_2沿染色体分布

本文以果蝇染色体的内含子和编码序列作为研究对象,用信息参数D2分析两类序列沿着染色体的分布规律,并用多项式拟合分析染色体各类序列沿染色体的统计分布特征,分析D2参数的平均值在不同的染色体臂的分布特征.

西藏牛Y染色体USP9Y基因与mtDNA D-loop区遗传多态性研究

本研究旨在探究西藏牛的Y染色体与mtDNA D-loop区的遗传多态性和起源。采用PCR扩增和限制性内切酶酶切的方法测定30头西藏牛Y染色体USP9Y基因的多态性;采用PCR扩增和测序技术测定30头西藏牛的mtDNAD-loop区序列,利用生物信息学的方法对西藏牛44条D-loop序列(其中14条从Gen Bank下载)进行遗传多态性分析。对30头西藏牛USP9Y基因的PCR产物进行电泳,共检测到471 bp和552 bp 2个片段,且552 bp片段不能被限制性内切酶Ssp I切开,说明西藏牛具有普通牛Y1和Y2 2种单倍型组,其频率分别为0.067和0.933。通过对44条西藏牛mtDNA D-loop全序列进行比对,发现本研究测定的30条西藏牛mtDNA D-loop全序列中,有8条为牦牛序列,说明西藏牛群体中有牦牛的基因渗入。对36条西藏牛mtDNA D-loop全序列进行分析,共检测到62个变异位点,界定了21个mtDNA单倍型,单倍型多样度为0.956 0,核苷酸多样度为0.006 4,表明西藏牛具有丰富的母系遗传多态性。系统发育树显示,西藏牛具有普通牛和瘤牛混合母系起源,且普通牛的单倍型在西藏牛群体中占绝对优势(95.24%)。西藏牛具有丰富的母系遗传多态性,由2个普通牛父系单倍型组构成,应归于普通牛一类,且群体中存在牦牛和瘤牛的基因渗入现象。

东亚族群中Y染色体D-M174单倍群的遗传多态性

目的研究Y染色体D-M174单倍群及下游分支在东亚族群的遗传多态性。方法收集东亚1426份无关男性样本,用微测序技术检测7个Y染色体D-M174单倍群及下游分支的Y-SNP位点,用DNATyperY26试剂盒检测22个Y-STR基因座,通过直接计数法、热图、系统发育聚类和网络图聚类进行单倍群分析,探讨Y染色体D单倍群遗传多态性分布、群体及样本间聚类关系。结果D-M174单倍群高频分布于西藏藏族人和日本人,分别为40.96%和35.71%,在西藏周边地区及其他地区呈现低频分布或无分布。结论Y染色体D-M174单倍群在东亚族群中具有明显的族群地域分布特征。

北江大刺鳅(Mastacembelus armatus)的核型分析及线粒体Cyt b基因和D-loop的遗传多样性

采用胸腔注射植物凝集素(PHA)、秋水仙素,以大刺鳅(Mastacembelus armatus)头肾组织为材料,低渗处理,空气干燥法进行染色体标本制备,分析其染色体核型。结果显示:大刺鳅的二倍体染色体数目为2n=48,其核型公式为2n=14m+34t, NF=62。对24尾大刺鳅线粒体Cyt b基因和D-loop序列进行测序分析,结果表明:(1)大刺鳅Cytb基因、D-loop序列长度分别为1438bp、895bp,两个基因的单倍型多样性分别为0.656和0.541,核苷酸多样性分别为0.00539和0.00817,平均核苷酸差异数分别为5.938和6.805,说明该种群的遗传多样性丰富。(2)两个基因的每个个体的遗传距离分别为0.000—0.018和0.000—0.026,说明该种群个体间的遗传距离小。(3)中性检验的Tajima’sD检验及Fu's Fs检验的结果说明该种群没有发生过扩张。结果说明北江大刺鳅的遗传多样性水平较高,可用于人工繁育。

从果蝇染色体DNA制备RNA探针及其染色体原位杂交定位

在相差显微镜下显微切割野生型黑腹果蝇唾液腺X染色体的1区片段,抽提该片段的DNA并与PGEM3Z质粒重组,然后转化到JM103 E.colt中,共切割12个染色体,抽提到大约10pgDNA,经重组、转化获得9个重组子。提取其中5个重组质粒分析其重组片段,2个约为0.6kb,另3个约为3.0kb。2个约为0.6kb重组片断用依赖DNA模板的SP6RNA聚合酶体外合成~3H标记的RNA,以此作探针进行染色体原位杂交定位,被克隆片段定位在果蝇唾液腺X染色体1E,1F位点上。本文结果表明重组到PGEM3Z质粒中染色体DNA可直接作为合成RNA探针的模板。

Amplification of D22S283 as a favorable prognostic indicator in liver fluke related cholangiocarcinoma

AIM： To analyze the DNA copy number of target genes NF2, TIMP3, ST13, TOB2, BIK, and TP and the reference microsatellite markers D22S283, D22S423, and D22S274 mapped on 22q12-qter in liver fluke related cholangiocarcinoma （CCA） and define its correlation with clinical parameters. METHODS： Quantitative real time PCR （qPCR） was used for determining allelic imbalances in 65 liver fluke related CCA tissues. Statistical correlations between allelic imbalances and clinicopathological parameters, i.e. age, sex, tumor stage, histological type, blood vessel invasion, nerve invasion and lymphatic invasion were evaluated by means of the X^2 test. Cox regression analysis was used for determining patient＇s survival. RESULTS： Amplifications of the TP （22q13.33）, TOB2 （22q13.2-13.31）, D22S283 （22q12.3）, TIMP3 （22q12.3） and NF2 （22q12.2） were found in 35 （53.8%）, 28 （43.1%）, 27 （41.5%）, 24 （36.9%）, and 24 （36.9%）, respectively. Losses at the D22S423 （22q13.1-13.2）and BIK （22q13.31） were detected in 26 （40%） and 23 （35.4%）, respectively. Significant correlations were observed between lymphatic invasion and allelic losses of BIK （P = 0.025） and D22S283 （P = 0.041）. Univariate and multivariate Cox regression analysis revealed D22S283 amplification as an independent predictor of good prognosis （P = 0.006, death hazard ratio = 0.411, 95% CI = 0.217-0.779） and blood vessel invasion as an independent poor prognostic factor （P = 0.042, death hazard ratio = 1.911, 95% CI = 1.022-3.571） in CCA patients. CONCLUSION： This study provides evidence for the involvement of gene amplification and deletion on chromosome 22q in liver fluke related CCA, This is the first report of D22S283 amplification as an independent indicator of favorable prognosis in liver fluke related CCA.

基于CMV启动子表达鹦鹉热衣原体PmpD-N蛋白的重组火鸡疱疹病毒的构建

为构建表达鹦鹉热衣原体多型外膜蛋白D的N端(PmpD-N)蛋白重组火鸡疱疹病毒(HVT),以鹦鹉热衣原体CB7株DNA为模板,通过PCR扩增pmpD-N基因,构建带有巨细胞病毒(CMV)启动子的真核表达盒,采用细菌人工染色体技术构建HVT重组子,转染细胞获得HVT重组病毒,并在体外评价该重组病毒的相关特性。结果表明,转染细胞后成功获得HVT重组病毒。通过Western blot和IFA分析表明,重组病毒表达了PmpD-N蛋白。Confocal分析表明,PmpD-N蛋白分布在细胞的胞核、胞浆和细胞表面。HVT重组病毒的复制特性和野生型HVT一致。HVT重组病毒能够激活巨噬细胞促炎性细胞因子IL-6和NO的产生。该试验成功构建了含有CMV启动子,能够表达PmpD-N蛋白的HVT重组病毒,为下一步进行动物试验和分析该病毒的免疫效力奠定了基础。

萝卜D染色体在7号连锁群的定位研究

在已知萝卜D染色体附加系对线虫具有抗性和萝卜分子图谱的7号连锁群定位有抗线虫基因Hs1Raph的基础上,利用(dp)RAPD分子标记技术,以甘蓝型油菜异源萝卜附加系为材料,筛选到90个萝卜D染色体的特异标记,选择4个在萝卜构图亲本间表现多态性的D染色体标记进行F2群体(包括245个单株)分离检测,并与构图用的505个AFLP和RAPD标记一起用Join-map3.0进行连锁定位分析。结果萝卜D染色体标记全部定位于7号连锁群,并且7号连锁群上只有D染色体的特异标记,说明萝卜7号连锁群就是D染色体在分子水平的反映。证实了定位有抗线虫基因Hs1Raph的7号连锁群对应于具线虫抗性效应的萝卜D染色体。实验结果对研究萝卜D染色体控制的农艺性状或抗逆性以及萝卜抗线虫育种和将萝卜抗线虫基因转入其它芸薹属作物的研究都将会起到重要的指导作用。

Effect of 2,4-D on Seedling Physiology and Cytogenetical Studies in Triticum aestivum and Phalaris minor (Gramineae)

Effects of 2,4-D on seedling growth and chromosomal abnormalities were studied in Triticum aestivum and Phalaris minor.Seeds were soaked at different concentrations of 2,4-D (0.01%,0.1%,1.0%) for 4,8,12 and 16 hours.2,4-D suppressed the germination more severely in P.minor than in T.aestivum.Shoot and root length was retarded with the increase of concentration and time of treatment in both species.Generally radical was more negatively affected than coleoptile and emergence of radical was not observed at 1.0% concentration at 8,12,and 16 hours of treatment in T.aestivum while in P.minor there was a total lack of radical emergence at 1.0% concentration for all durations of treatment.Stiff and curled roots and undifferentiated callus like scutellar tissues were observed in T.aestivum,while in P.minor the coleoptile obtained was lean,pale green in colour and was lying flat on filter paper.Mitotic index decreased,while chromosomal abnormalities,bridges and laggards were increased with the increase of concentration and soaking time however,laggards were not observed in T.aestivum.Clumping and chain formation of chromosomes at metaphase was also noticed in P.minor.

Large-scale data-driven and physics-based models offer insights into the relationships among the structures,dynamics,and functions of chromosomes

The organized three-dimensional chromosome architecture in the cell nucleus provides scaffolding for precise regulation of gene expression.When the cell changes its identity in the cell-fate decision-making process,extensive rearrangements of chromo-some structures occur accompanied by large-scale adaptations of gene expression,underscoring the importance of chromosome dynamics in shaping genome function.Over the last two decades,rapid development of experimental methods has provided unprecedented data to characterize the hierarchical structures and dynamic properties of chromosomes.In parallel,these enormous data offer valuable opportunities for developing quantitative computational models.Here,we review a variety of large-scale polymer models developed to investigate the structures and dynamics of chromosomes.Different from the underlying modeling strategies,these approaches can be classified into data-driven(‘top-down’)and physics-based(‘bottom-up’)categories.We discuss their contributions to offering valuable insights into the relationships among the structures,dynamics,and functions of chromosomes and propose the perspective of developing data integration approaches from different experimental technologies and multidisciplinary theoretical/simulation methods combined with different modeling strategies.

Inter3D:Capture of TAD Reorganization Endows Variant Patterns of Gene Transcription

Topologically associating domain(TAD)reorganization commonly occurs in the cell nucleus and contributes to gene activation and inhibition through the separation or fusion of adjacent TADs.However,functional genes impacted by TAD alteration and the underlying mechanism of TAD reorganization regulating gene transcription remain to be fully elucidated.Here,we first developed a novel approach termed Inter3D to specifically identify genes regulated by TAD reorganization.Our study revealed that the segregation of TADs led to the disruption of intrachromosomal looping at the myosin light chain 12B(MYL12B)locus,via the meticulous reorganization of TADs mediating epigenomic landscapes within tumor cells,thereby exhibiting a significant correlation with the down-regulation of its transcriptional activity.Conversely,the fusion of TADs facilitated intrachromosomal interactions,suggesting a potential association with the activation of cytochrome P450 family 27 subfamily B member 1(CYP27B1).Our study provides comprehensive insight into the capture of TAD rearrangement-mediated gene loci and moves toward understanding the functional role of TAD reorganization in gene transcription.The Inter3D pipeline developed in this study is freely available at https://github.com/bm2-lab/inter3D and https://ngdc.cncb.ac.cn/biocode/tool/BT7399.

蛋白质分散的纳米红色元素硒的延缓衰老作用

目的 考察以蛋白质为核、红色元素硒为膜和以蛋白质为分散剂的纳米红色元素硒是否具有延缓衰老作用。方法 采用 D-半乳糖小鼠衰老和黑腹果蝇生存模型 ,评价纳米红色元素硒的抗氧化和延长生存时间作用。结果 纳米红色元素硒能显著降低小鼠全血丙二醛含量和提高小鼠全血谷胱甘肽过氧化物酶活性 ,显著延长黑腹果蝇生存时间。

蜜环菌多糖对低能离子束诱变作用的恢复效应

水提法从蜜环菌 Am 99- 1菌株人工发酵菌体分离得到蜜环菌多糖 Am 1,以低能 N+ 离子束为诱变源 ,黑腹果蝇为受试动物 ,检测 Am 1对离子束诱变果蝇的恢复功能。实验结果经统计学分析表明 ,1.0 %和 1.5 %浓度的蜜环菌多糖 Am 1可以提高受试果蝇蛹的孵出率和降低当代及 F2代果蝇突变率。这说明蜜环菌多糖对于低能离子束诱变有一定程度的防护作用。

果蝇(Drosophila melanogaster)唾腺染色体的制备及染色体识别

果蝇(Drosophila melanogaster)唾腺染色体的制备与分析是果蝇细胞遗传研究的基本实验技术,也是非常流行的遗传学教学实验。国内绝大多数的实验教材介绍了这个实验的操作方法,但对于如何辨别染色体各条臂,并进而识别具体的带纹均未予以介绍。笔者详细介绍唾腺染色体的制备及拍摄、数码照片拼接方法;并着重介绍如何根据各条臂端粒端的形态特征,以及利用Lefevre的唾腺染色体照片图谱中指示的蓬突(puff)、缢缩及带纹组合特征识别染色体臂的方法,供研究和实验教学参考。