蛋白质Sumo化修饰原核表达系统的构建

【目的】构建一种可获得Sumo修饰蛋白的原核表达系统,为Sumo化修饰蛋白的批量制备奠定基础。【方法】用Trizol裂解法提取小鼠畸胎瘤细胞F9的总RNA,反转录成cDNA后PCR扩增获得Sumo1、Ubc9、Sae2和Sae1基因。将上述基因及相应载体酶切、连接后构建出重组质粒pACYC-Ubc9-Sumo1、pCDF-Sae2-Sae1。采用质粒转化制备阳性克隆感受态细胞质粒转化的方法获得pACYC-Ubc9-Sumo1和pCDF-Sae2-Sae1共表达的Sumo化修饰的原核表达系统。以pGEX-GST-Sox2和pGEX-GST-Sox2^K247R重组质粒为例,采用Western Blot检测蛋白质Sumo化修饰原核表达系统的有效性。用另一Sumo修饰底物Oct4及其Sumo修饰位点突变的突变体Oct4^K118R转化原核Sumo修饰系统,并连续传代,检测蛋白质Sumo化修饰系统的实用性及遗传稳定性。【结果】构建的蛋白质Sumo化修饰原核表达系统可特异修饰Sumo1的底物Sox2和Oct4;含有Oct4原核表达载体的蛋白质Sumo化修饰系统可稳定传至15代,系统的稳定性较好。【结论】获得了成本较低、特异性强、重复性好、稳定性强的可用于制备Sumo化蛋白的原核表达系统。     

Influence of uterine site on occurrence of spontaneous cleft lip in mice

It has been found that within the A/Jax strain, embryos in the uterine site nearest the ovary develop cleft lip (with or without cleft palate) significantly more often than embryos in other positions in the uterus.     

Induction of tumors in mice by genomic hypomethylation

Genome-wide DNA hypomethylation occurs in many human cancers, but whether this epigenetic change is a cause or consequence of tumorigenesis has been unclear. To explore this phenomenon, we generated mice carrying a hypomorphic DNA methyltransferase 1 (Dnmt1) allele, which reduces Dnmt1 expression to 10% of wild-type levels and results in substantial genome-wide hypomethylation in all tissues. The mutant mice were runted at birth, and at 4 to 8 months of age they developed aggressive T cell lymphomas that displayed a high frequency of chromosome 15 trisomy. These results indicate that DNA hypomethylation plays a causal role in tumor formation, possibly by promoting chromosomal instability.     

苎麻谷氨酰胺合成酶BnGS2等位基因的克隆及其转基因烟草特性

【目的】克隆谷氨酰胺合成酶BnGS2等位基因,分别构建其超量表达载体,并探讨其在转基因烟草中对氮代谢的影响。【方法】依据苎麻转录组unigenes和RT-PCR技术克隆苎麻BnGS2等位基因,利用内切酶TaqⅠ对目的等位基因在中苎1号自交F1和亲本中进行酶切鉴定,并利用生物信息学对基因序列和结构特征进行分析;通过同源重组技术分别构建BnGS2等位基因的超量表达载体,并在农杆菌（Agrobacterium tumefaciens）LBA4404的介导下,通过叶盘法将超量表达载体转入烟草中,通过Kan筛选、转化植株基因组DNA PCR验证获得转基因T0植株;利用qRT-PCR分析BnGS2等位基因在转基因T1植株中的相对表达水平,并测定植株叶片中的GS活性、株高、鲜重、可溶蛋白及总氮含量。【结果】首次从苎麻中克隆了一对GS2等位基因,命名为BnGS2-1和BnGS2-2,等位基因序列全长1 340 bp,含有一个1 293 bp的ORF区,编码430个氨基酸残基多肽;等位基因核苷酸序列在11个位点上存在差异,导致编码的多肽在195、382位点上的氨基酸残基存在替换现象（BnGS2-1为脯氨酸和天冬酰胺,BnGS2-2为苏氨酸和丝氨酸）;NCBI BLASTP分析表明苎麻BnGS2与Pisum sativum、Vigna radiata、Glycine max、Phaseolus vulgaris、Medicago truncatula具有很近的亲缘关系;构建了能分别超量表达BnGS2-1和BnGS2-2的载体,并获得能分别超量表达BnGS2-1和BnGS2-2转基因烟草植株;与野生型烟草植株相比,超量表达BnGS2等位基因（BnGS2-1和BnGS2-2）都能显著性提高转基因植株叶片GS活性、鲜重和可溶性蛋白的含量,株高和总氮含量也有增加,但没有达到显著性水平。另外,超量表达不同BnGS2等位基因（BnGS2-1和BnGS2-2）的转基因烟草植株,在株高、鲜重、叶片可溶性蛋白及总氮含量上并没有显著性差异。【结论】在烟草中分别超量表达来源苎麻的BnGS2等位基因（BnGS2-1或BnGS2-2）均能显著提高转基因植株的生物产量和氮利用效率,并且所克隆的2个等位基因BnGS2-1和BnGS2-2在功能上并无显著差异。     

鳄鱼源气单胞菌的毒力基因与耐药特性分析

对海南地区患病暹罗鳄(Crocodylus siamensis)体内分离出的10株气单胞菌(5株嗜水气单胞菌、3株达卡气单胞菌、1株简达气单胞菌和1株维氏气单胞菌)进行了毒力基因、耐药基因的检测以及抗生素药物敏感性试验.结果 表明:气溶素、肠毒素、蛋白酶等基因检出率较高,其中,lip和ela2种毒力基因检出率高达100...     

两种国兰MADS-box家族B类基因PI/GLO的克隆与时空表达特性分析

为了挖掘国兰PISTILLATA/GLOBOSA（PI/GLO）基因的功能,利用生物信息学方法对两种国兰PI/GLO进行鉴定分析,并利用实时荧光定量的方法对其表达模式进行分析。结果表明,所克隆的两个 PI/GLO均为MIKC型MADS-box基因,均有保守的MADS区和相对保守的K区,且与春兰（AD158461.1）同源性较高,分别为98%、99%。两个基因的登录号为：MW654194,MW654193。RT-qPCR试验分析得出, CfPI1 在蕙兰各组织器官中的相对表达丰度顺次为：花萼>花瓣>唇瓣>花蕾>子房>盛花期花葶>合蕊柱, CsPI1 在墨兰中的相对表达丰度顺次为：花瓣>唇瓣>花蕾>花萼>合蕊柱>子房。两个基因均在盛花期的表达远远高于花蕾期和营养生长期,且主要参与花器官的生长发育。     

Identification of additional QTLs for flowering time by removing the effect of the maturity gene E1 in soybean

The adaptability of soybean to be grown at a wide range of latitudes is attributed to natural variation in the major genes and quantitative trait loci(QTLs) that control flowering time and maturity. Thus, the identification of genes controlling flowering time and maturity and the understanding of their molecular basis are critical for improving soybean productivity. However, due to the great effect of the major maturity gene E1 on flowering time, it is difficult to detect other small-effect QTLs. In this study, aiming to reduce the effect of the QTL, associated with the E1 gene, on the detection of other QTLs, we divided a population of 96 recombinant inbred lines(RILs) into two sub-populations: one with the E1 allele and another with the e1 nl allele. Compared with the results of using all 96 recombinant inbred lines, additional QTLs for flowering time were identified in the sub-populations, two(q FT-B1 and q FT-H) in RILs with the E1 allele and one(q FT-J-2) in the RILs with the e1 nl allele, respectively. The three QTLs, q FT-B1, q FT-H and q FT-J-2 were true QTLs and played an important role in the regulation of growth period. Our data provides valuable information for the genetic mapping and gene cloning of traits controlling flowering time and maturity and will help a better understanding of the mechanism of photoperiod-regulated flowering and molecular breeding in soybean.     

2个低直链淀粉含量籼稻突变体的遗传分析

对2个空间诱变低直链淀粉含量籼稻突变体XLA-1和XLA-2进行了遗传分析和分子生物学研究.结果表明:XLA-1低直链淀粉遗传特性受2对隐性基因控制,这2对基因具有连锁关系和互补作用,任何1对基因隐性纯合都将导致直链淀粉含量降低;XLA-2低直链淀粉遗传特性受1对隐性主效基因控制,该基因可能为Wx的等位基因,同时受微效基因的修饰.XLA-1和XLA-2蜡质基因(CT)n微卫星多态性与糯稻相同,但其直链淀粉质量分数分别为14.42%和11.59%,说明除Wx基因外确实还有其他影响直链淀粉含量的遗传因素,这与遗传分析结果相符.     

瑞华麦523PPO活性及1BL/1RS易位相关基因的分子检测

【目的】了解小麦品种瑞华麦523及其亲本郑麦9023和烟1604的PPO和1BL/1RS易位相关基因的遗传信息,旨在提高优质小麦品种选育的效率。【方法】利用小麦PPO活性基因的分子标记PPO18、PP0-B5、PPO16和PPO29及1BL/1RS易位的分子标记H20,检测小麦品种瑞华麦523及其亲本相关基因的等位变异及来源。【结果】小麦品种瑞华麦523在2AL染色体上PPO等位基因为PPO-A1a基因型,与亲本郑麦9023一样,为高活性PPO,不含亲本烟1604的低活性PPO;在2BL染色体上PPO等位基因为A型,与亲本一致,是高活性PPO;在2DL染色体上PPO等位基因为PPO-D1b基因型,与亲本郑麦9023一样,是一个高活性PPO,不含亲本烟1604的低活性PPO;利用分子标记H20检测表明,瑞华麦523和亲本郑麦9023一样,是一个非1BL/1RS易位系。【结论】瑞华麦523是一个具有高活性PPO、非1BL/1RS易位系的小麦品种,其小麦PPO和1BL/1RS易位相关基因主要来自母本郑麦9023,为该品种的推广应用和培育优质小麦新品种提供理论参考。     

Molecular biology. Mothers setting boundaries

Certain genes are only expressed at one allele, a phenomenon called imprinting. Although it is well established that one allele of certain imprinted genes is silenced through methylation, this does not appear to be the case for all imprinted genes. In a thoughtful Perspective, Thorvaldsen and Bartolomei discuss new findings showing that insertion of insulator elements (boundary regions) between the promoter of a gene and its enhancer (a sequence that boosts gene expression) may be another way in which genes are silenced during imprinting.     

Allelic Variation in Loci for Adaptive Response and Its Effect on Agronomical Traits in Chinese Wheat (Triticum aestivum L.)

Heading date was an important trait that decided the adaptation of wheat to environments. It was modified by genes involved in vernalization response, photoperiod response and development rate. In this study, four loci Xgwm261, Xgwm219, Xbarc23 and Ppd-D1 which were previously reported related to heading time were analyzed based on three groups of wheat including landraces (L), varieties bred before 1983 (B82) and after 1983 (A83) collected from Chinese wheat growing areas. Generally, heading date of landrace was longer than that of varieties. Significant differences in the heading time existed within the groups, which implied that diversification selection was much helpful for adaptation in each wheat zone. Photoperiod insensitive allele Ppd-D1a was the first choice for both landrace and modern varieties, which promoted the heading date about four days earlier than that of sensitive allele Ppd-D1b. The three SSR loci had different characters in the three groups. Predominant allele combination for each zone was predicted for wheat group L and A83, which made great contribution to advantageous traits. Xgwm219 was found to be significantly associated with heading date in Yellow and Huai River Winter Wheat Zone (Zone II) and spike length in Middle and lower Yangtze Valley Winter Wheat Zone (Zone III), which implied functional diversification for adaption. Variation for earliness genes provided here will be helpful for whet breeding in future climatic change.     

两个中国小麦品种中抗叶锈基因的遗传分析和基因定位

【目的】确定来自四川的两个小麦品种绵阳351-15和SW8588所携带的抗叶锈基因,为选育持久抗锈品种提供理论依据。【方法】在苗期用15个叶锈菌生理小种接种小麦品种绵阳351-15、SW8588和30个含有已知抗叶锈基因的近等基因系,推导2个材料中所含有的抗叶锈病基因,同时以小麦抗叶锈品种绵阳351-15和SW8588分别同感病品种郑州5389杂交获得F1和F2代群体,用叶锈菌小种FHTT接种各亲本及其杂交后代,进行抗叶锈遗传分析,并利用SSR和STS标记进行抗叶锈病基因的分子定位。【结果】经苗期基因推导发现SW8588中含有未知基因不同于已知抗叶锈病基因Lr1,绵阳351-15中可能含有已知抗叶锈病基因Lr1。用叶锈菌小种FHTT接种各F1和F2代群体,2个F2代群体抗感单株分离比例均符合3﹕1的理论分离比例,表明2个亲本对小种FHTT的抗病性均由1个显性基因控制。经过分子标记分析,在小麦材料绵阳351-15中发现该抗叶锈基因与位于5DL的SSR标记barc144和wmc765连锁,其遗传距离分别为8.9和20.8 cM,并同Lr1的STS标记WR003共分离,确定在小麦材料绵阳351-15中对小种FHTT的抗病性由抗叶锈基因Lr1提供;经分子标记检测SW8588中含有1对显性的抗叶锈病基因,暂命名为LrSW85,该基因位于5DL染色体上与Lr1的STS标记WR003共分离,该抗叶锈基因可能是Lr1的等位基因或紧密连锁基因。【结论】通过基因推导、遗传分析和分子标记等手段,确定小麦材料绵阳351-15中含有抗叶锈基因Lr1;小麦材料SW8588中含有抗叶锈基因LrSW85,该基因可能为Lr1的等位基因或紧密连锁基因。     

部分新疆小麦材料多酚氧化酶活性基因分布规律研究

[目的]了解新疆小麦材料多酚氧化酶(PPO)活性基因的等位变异组成和分布以及与PPO活性的关系,为小麦PPO活性的分子标记辅助选择奠定基础.[方法]利用PPO基因的功能标记PPO18、PPO29和PPO16对445份新疆冬春小麦材料进行2A和2D染色体上PPO等位基因Ppo -A1a、Ppo - A1b、Ppo - D1a和Ppo - D1b的检测,分析不同等位变异与PPO活性的相关性及变化趋势.[结果]PPO18在等位基因Ppo-A1a(高PPO)和Ppo - A1b(低PPO)中的分布频率为73.03;和26.97;,PPO16和PPO29在等位基因Ppo-D1a(低PPO)和Ppo - D1b(高PPO)中的分布频率76.63;和23.37;.Ppo -A1a与Ppo -A1b基因型的PPO活性平均值差异达极显著水平(P＜0.01).而Ppo- D1a与Ppo - D1b两者基因型的平均PPO活性差异不显著(P0.05);两个PPO基因的等位基因组合类型分布频率为:Ppo -A1a/Ppo - D1a(54.38;)、Ppo - A1a/Ppo-D1b( 18.65;)、Ppo -A1b/Ppo - D1a(22.25;)和Ppo -A1b/Ppo -D1b(4.72;);同时冬春小麦材料间PPO活性基因分布存在明显差异,总体来看,新疆小麦材料中高PPO活性的等位变异类型所占比例较高.[结论]PPO18、PPO16和PPO29标记可以作为小麦材料PPO活性鉴定有效工具.可直接利用此标记进行标记辅助选择.     

利用STS标记检测CIMMYT小麦品种（系）中Lr34/Yr18、Rht-B1b和Rht-D1b基因的分布

 【目的】明确慢锈基因Lr34/Yr18和矮秆基因Rht-B1b（Rht-1）、Rht-D1b（Rht-2）在CIMMYT小麦品种中的分布,帮助慢病性品种选育和株高改良。【方法】使用3个STS标记,检测慢锈基因Lr34/Yr18和矮秆基因Rht-B1b、Rht-D1b在263个CIMMYT小麦品种和高代品系中的分布。【结果】csLV34标记在含Lr34/Yr18的材料中扩增出一条150bp的特异带,在不含Lr34/Yr18的材料中扩增出229bp的特异带;利用2对互补引物NH-BF.2/WR1.2和NH-BF.2/MR1对Rht-B1a和Rht-B1b基因进行检测,在携带Rht-B1a和Rht-B1b的材料中分别扩增出一条400 bp的特异带;利用DF/MR2标记检测Rht-D1b基因,在携带Rht-D1b的材料中,扩增出一条280 bp的片段。在263个品种中,57个品种携带Lr34/Yr18基因,占总数的21.7%;216个品种含Rht-B1b,占总数的82.1%;38个品种含Rht-D1b,占总数的14.4%。含双矮秆基因型（Rht-B1b+Rht-D1b）的品种有12个,不含这2个矮秆基因的品种（Rht-B1a+Rht-D1a）有21个。【结论】这3个STS标记可以方便、快速、准确地检测Lr34/Yr18、Rht-B1b和 Rht-D1b基因。在CIMMYT材料中,Rht-B1b基因频率很高,Rht-D1b较低。     

蝴蝶兰PhaSEP3基因的克隆及其在突变体中的表达

E类MADS-box是花器官发育分子模型中必不可少的基因,通过突变体研究其表达模式将为深入理解兰科植物花器官的分子机理与完善花发育调控理论提供依据。采用RACE(rapid-amplification of cDNA ends)技术从蝴蝶兰花瓣中克隆了一个E类MADS-box基因PhaSEP3(GenBank登录号为MZ436812),并采用实时荧光定量PCR(qRT-PCR)分析了该基因在蝴蝶兰不同组织和5种突变体中的表达水平。结果表明,该基因cDNA全长为1 236 bp,具有753 bp的开放阅读框(ORF),可编码250个氨基酸,其C端具有SEPⅠ和SEPⅡ基序。系统进化分析显示,该基因编码蛋白质与蝴蝶兰属的PeSEP3和AGL9亲缘关系最近。组织特异表达分析表明,PhaSEP3基因主要在生殖器官和授粉后子房中表达;在不同突变体中,PhaSEP3基因在侧萼唇瓣化突变体的萼片和唇瓣中表达水平显著升高;在退化雄蕊瓣化突变体的侧瓣、唇瓣和子房中表达水平显著降低,在蕊柱中的表达水平显著升高;在侧瓣唇瓣化突变体的侧瓣和唇瓣中表达水平显著升高;在侧瓣退化突变体的侧瓣中表达水平显著降低,而在蕊柱和子房中表达水平显著升高;在侧瓣雄化突变体中,该基因的表达水平在萼片和侧瓣中均显著升高。分析认为,PhaSEP3基因主要调控蝴蝶兰花器官各轮组织与授粉后子房的发育,在突变体花器官中,PhaSEP3类基因可能与其他花发育基因互作参与花器官形态的发育调控。该研究结果为进一步理解兰科植物花器官多样性的调控机理提供了资料。     

Insecticidal activity and lectin homology of arcelin seed protein

Arcelin, a major seed protein discovered in wild beans (Phaseolus vulgaris), has toxic effects on an important bean bruchid pest, Zabrotes subfasciatus. Transfer of the arcelin-1 allele to bean cultivars and addition of purified arcelin to artificial seeds results in high levels of insect resistance. The nucleotide and derived amino acid sequences of the arcelin-1 complementary DNA are very similar to those of genes encoding the bean seed lectin, phytohemagglutinin. The gene or genes encoding arcelin may have evolved from a phytohemagglutinin gene or genes resulting in an effective mechanism for resistance to bean bruchids.     

滇I型杂交粳稻DH群体四个数量性状的遗传分析

以滇Ⅰ型杂交粳稻 (寻A×南 2 9- 2 )F1代花药培养的DH群体共 6 7系为材料 ,分析了穗长、一次枝梗数、穗颈长、实粒数 4个性状的遗传力和基因对数 ,4个性状的遗传力分别为 :0 70 16 ,0 4 0 87,0 75 0 3,0 5 86 1,基因对数分别为 :6 835 ,2 3 2 0 4 ,8 94 7,17 36 7对。通过穗长、一次枝梗数、穗颈长、实粒数 4个性状的偏度和峰度系数分析 ,发现 :控制穗长的多基因间无互作 ;控制一次枝梗数、穗颈长的多基因间可能存在互补作用 ,但把一次枝梗数分解成 3个等级后 ,即 1级为主穗一次枝梗数 ,2级为第 1分蘖的一次枝梗数 ,3级为第 2分蘖的 2次枝梗数 ,遗传力分别是 :0 2 0 6 0 ,0 2 0 4 6 ,0 135 6 ,基因对数分别为 :2 1 90 5 ,2 6 0 4 5 6 ,38 2 883对 ,基因间都无互补作用 ;影响实粒数的多基因间存在互补作用。     

小麦雄性核不育基因Tal与rht_(10)基因的连锁遗传

几年来,进行了普通小麦显性雄性核不育基因Tal与矮变1号的矮秆基因Rht_(1c)的等位基因rht_(10)的连锁遗传,以及试图打破其连锁方式,选育重组类型Tal-Rhhrht_(10),使Tal转育至矮变1号的研究,这项研究目前只能获得非期望的另一类tal-rht_(10)交换型配子,本研究表明,两个基因有复杂的连锁关系,可能Tal不仅与rht_(10)主基因,而且与某个修饰基因连锁,重组型是致死的。