酵母蛋白激酶Sch9激活环磷酸化状态参与热胁迫应答调控

在酵母(Saccharomyces cerevisiae)中,蛋白激酶Sch9已经被证明与细胞大小、胁迫应答、自噬以及寿命的调控有关.通过不同基因型酵母在固体培养基上的热敏感性研究,发现Sch9的激酶功能与热胁迫相关,进一步通过Sch9特异磷酸化位点抗体的免疫印迹,检测了细胞内Sch9关键的激活环T570位点(PDK1位点)在热胁迫条下的磷酸化状态,发现半乳糖诱导表达的Sch9在热胁迫条件下T570位点去磷酸化,首次证明了Sch9通过调控激酶活性参与细胞热胁迫应答.研究结果揭示了Sch9在胁迫应答中的部分功能,并为生理条件下Sch9调控功能的研究提供了生物化学证据.     

甘蔗泛素结合酶基因的克隆与表达北大核心CSCD

为克隆鉴定甘蔗泛素结合酶基因（Sugarcane ubiquitin-conjugating enzyme,ScUBc E2）并探索其在激素信号通路和甘蔗与黑穗病互作过程中的作用,选择黑穗病菌胁迫下甘蔗抑制消减杂交文库（Suppression subtractive hybridization,SSH）中注释为泛素结合酶的差异表达EST序列为探针,结合电子克隆技术和RT-PCR技术,以甘蔗cDNA为模板进行泛素结合酶基因克隆.对克隆获得的序列进行生物信息学分析,并利用qRT-PCR技术分析该基因在甘蔗根、蔗髓、叶、芽中的组织特异性表达以及在黑穗病菌、茉莉酸甲酯（Methyl jasmonate,Me JA）、脱落酸（Abscisic acid,ABA）和水杨酸（Salicylic acid,SA）胁迫下的表达情况.最终克隆得到一条长度为699 bp的甘蔗泛素结合酶基因（ScUBc E2;Gen Bank accession number：KJ577594.1）,该基因包含长度为447 bp的完整开放读码框,编码148个氨基酸.生物信息学分析结果显示,ScUBc E2编码的蛋白分子量（Mr）为16.507×10^3;无信号肽,为碱性不稳定的亲水蛋白;包含4个α螺旋、4个β折叠和一些无规则卷曲;第15位氨基酸为泛素化位点,74-89位氨基酸为活性位点;在进化过程中与高粱泛素结合酶基因的亲缘关系最近.qRT-PCR分析结果表明,ScUBc E2基因组成型表达,但在芽中的表达量最高;其表达在甘蔗感黑穗病基因型ROC22中受到黑穗病菌胁迫的抑制,在黑穗病抗病基因型YC05-179中则先被抑制,后被诱导;ScUBc E2基因受MeJA及SA诱导表达,对ABA胁迫的响应不明显.本研究表明,甘蔗ScUBc E2基因在抗病基因型和感病基因型甘蔗中存在不同表达模式,可能参与甘蔗与黑穗病菌的互作过程,有望为抗病育种分子标记提供潜在基因资源;同时,该基因受MeJA和SA外源激素胁迫后的表达模式,可为泛素-蛋白酶体途径及激素调控的信号转导在甘蔗与黑穗病菌互作过程中的作用提供一定的理论依据.     

甘蔗泛素结合酶基因的克隆与表达

为克隆鉴定甘蔗泛素结合酶基因(Sugarcane ubiquitin-conjugating enzyme,ScUBc E2)并探索其在激素信号通路和甘蔗与黑穗病互作过程中的作用,选择黑穗病菌胁迫下甘蔗抑制消减杂交文库(Suppression subtractive hybridization,SSH)中注释为泛素结合酶的差异表达EST序列为探针,结合电子克隆技术和RT-PCR技术,以甘蔗cDNA为模板进行泛素结合酶基因克隆.对克隆获得的序列进行生物信息学分析,并利用qRT-PCR技术分析该基因在甘蔗根、蔗髓、叶、芽中的组织特异性表达以及在黑穗病菌、茉莉酸甲酯(Methyl jasmonate,Me JA)、脱落酸(Abscisic acid,ABA)和水杨酸(Salicylic acid,SA)胁迫下的表达情况.最终克隆得到一条长度为699 bp的甘蔗泛素结合酶基因(ScUBc E2;Gen Bank accession number:KJ577594.1),该基因包含长度为447 bp的完整开放读码框,编码148个氨基酸.生物信息学分析结果显示,ScUBc E2编码的蛋白分子量(Mr)为16.507×10~3;无信号肽,为碱性不稳定的亲水蛋白;包含4个α螺旋、4个β折叠和一些无规则卷曲;第15位氨基酸为泛素化位点,74-89位氨基酸为活性位点;在进化过程中与高粱泛素结合酶基因的亲缘关系最近.qRT-PCR分析结果表明,ScUBc E2基因组成型表达,但在芽中的表达量最高;其表达在甘蔗感黑穗病基因型ROC22中受到黑穗病菌胁迫的抑制,在黑穗病抗病基因型YC05-179中则先被抑制,后被诱导;ScUBc E2基因受MeJA及SA诱导表达,对ABA胁迫的响应不明显.本研究表明,甘蔗ScUBc E2基因在抗病基因型和感病基因型甘蔗中存在不同表达模式,可能参与甘蔗与黑穗病菌的互作过程,有望为抗病育种分子标记提供潜在基因资源;同时,该基因受MeJA和SA外源激素胁迫后的表达模式,可为泛素-蛋白酶体途径及激素调控的信号转导在甘蔗与黑穗病菌互作过程中的作用提供一定的理论依据.     

细胞絮凝及硫酸锌对酿酒酵母乙酸胁迫耐性的影响

酿酒酵母细胞絮凝和外源添加锌离子对其环境胁迫耐受性都具有促进作用,为了解细胞絮凝形态对锌促进乙醇发酵的影响,比较硫酸锌添加对絮凝酿酒酵母SP SC01及其絮凝基因失活突变体SPSC01 FLO1Δ在乙酸胁迫条件下乙醇发酵的影响.结果显示,与野生型絮凝酵母相比,添加锌可更明显改善SPSC01 FLO1Δ在乙酸中的生长和发酵,在10 g L~(-1)乙酸存在的情况下,SPSC01在70 h消耗100 g L~(-1)葡萄糖,锌离子添加后可使发酵终点提前10 h,而SPSC01FLO1Δ在锌离子添加后发酵时间为48 h,可将发酵时间显著缩短138 h.这些结果表明,锌在酿酒酵母细胞缺少絮凝保护的条件下更能有效发挥作用,同时甘油、琥珀酸的增加在絮凝基因敲除突变体中更加明显.本文研究结果可为进一步利用絮凝及锌响应调控基因提高酿酒酵母的环境胁迫耐受性,提高燃料乙醇的生产效率奠定基础.     

Dietzia菌随机插入突变体文库的构建及功能基因筛选

Dietzia属菌具有很好的烃类降解和良好的高盐、高碱耐受能力.为研究Dietzia属降解烷烃和耐受盐碱的分子机制,以Dietzia sp. DQ12-45-1b为研究菌株,利用双质粒系统p Tip-istAB-sacB和pRTSK-sacB,成功构建库容为30 720的随机突变体文库.随机挑选的突变克隆及突变克隆传代20次后的Southern blot结果表明:转座序列插入的随机性较好,均为单插入突变,并且具有较好的遗传稳定性,可用于长期筛选功能基因.在此基础上,分别以高盐培养和正十六烷为单一碳源培养,利用高通量基因筛选,成功获得了一个耐盐突变体M9G和5个降解利用正十六烷相关突变株M6A、M6B、M7A、M9A、M81C.与野生型相比,5个降解利用正十六烷相关突变株的降解率下降范围为16.67%-84.35%.插入位点分析结果显示,M9G、M6A、M6B、M7A、M9A和M81C的插入基因分别预测为丝氨酸蛋白酶、铁氧化还原蛋白、假定蛋白、铁氧化还原蛋白还原酶、ABC转运蛋白和细胞色素P450类CYP153烷烃羟化酶.其中除了预测假定蛋白外,有些基因与传统上确定的功能存在差异,暗示着其可能具有新的功能.Dietzia菌随机插入突变体文库的构建及筛选可为研究Dietzia菌的抗逆和烷烃降解机制奠定基础.(图6表3参28)     

深海细菌Celeribacter indicus P73~T对菲的降解机制

来自深海环境的多环芳烃降解菌Celeribacter indicus P73~T能够高效降解菲,为揭示菲生物降解的分子机制,对其降解途径进行分析.通过GC-MS联用技术鉴定出菌株P73~T降解菲的2个重要的中间代谢产物,1-羟基-2-萘甲酸和1-萘酚.通过分析菌株P73~T全基因组,发现了菲降解基因簇(P73_0346-P73_0354),编码包括环羟基化双加氧酶、二氢二醇脱氢酶、环裂解双加氧酶、异构酶、水合醛缩酶等.通过验证环羟基化双加氧酶大亚基基因突变株ΔP73_0346::kan的菲降解能力,证实基因P73_0346编码了菲双加氧酶.依据代谢物检测、基因组分析和突变株功能验证结果,推测菌株P73~T降解菲经由菲C3,4-双加氧途径,更进一步地确定了参与此途径的菲双加氧酶等降解相关基因.本研究不仅揭示了菲降解的分子机制,也为菲污染的生物修复技术提供了理论依据.     

睾丸酮丛毛单胞菌teiR基因插入失活突变体构建及降解特性分析半

睾丸酮丛毛单胞菌(Comamonas testosteroni)是革兰氏阴性菌,能够以各种类固醇化合物作为唯一碳源和能源,生物信息学分析发现睾丸酮丛毛单胞菌teiR基因编码蛋白属于LuxR-type转录调控蛋白.利用同源重组原理构建teiR基因插入失活突变体C. mut,初步研究TteiR基因在类同醇代谢途径中的作用,以及对睾丸酮丛毛单胞菌关键降解酶3a-HSD/CR的表达影响.结果发现,teiR基因插入失活突变体C.mut在LB培养基中的生长速率不受影响,突变体C.mut抑制了teiR基因和关键降解酶3a-HSD/CR的表达,同时突变体C. mut丧失了以睾丸酮作为碳源进行生长的能力.结果表明teiR基因调节睾丸酮丛毛单胞菌类因醇代谢,同时teiR基因表达对3a-HSD/CR基因的表达是必需的.图7参14     

利用CRISPR/Cas9技术编辑水稻负调控抗病基因OsEDR1及基因功能分析

拟南芥EDR1(Enhanced Disease Resistance1),一个Raf样MAPKKK蛋白激酶,通过级联蛋白激酶途径MKK4/MKK5-MPK3/MPK6负调控水杨酸诱导的免疫防卫反应.为研究水稻OsEDR1基因的功能,利用CRISPR/Cas9技术创制OsEDR1的功能缺失突变体的同时,构建过表达载体p TCK303-Ubi-OsEDR1转化水稻,获得转基因植株,最后接种白叶枯病菌.此外,也构建pBI1.4t-35S-OsEDR1表达载体并转化拟南芥,分析转基因植株的白粉菌抗病性.结果表明利用CRISPR/Cas9技术创制了单碱基插入的OsEDR1功能缺失突变体;白叶枯病原菌Xoo PXO99的接种实验表明,该功能缺失突变体与日本晴相比,病斑长度减少约50%,增强了水稻对Xoo PXO99的抗性.定量PCR和接种实验结果显示在日本晴中过量表达OsEDR1增强了水稻对Xoo PXO86的感病性.在拟南芥edr1突变体中异源表达OsEDR1无法抑制edr1突变体对白粉菌的抗病和白粉菌诱导的细胞死亡.综上所述,OsEDR1负调控水稻对白叶枯的抗病反应和自发的细胞死亡,可能与拟南芥所依赖的防御反应信号通路不同相关;结果可为下一步深入研究EDR1基因的作用机制和在水稻育种中的应用提供参考.(图4表1参29)     

不同根瘤菌中突变cysDN基因对硫酸盐同化途径的影响

硫是生物体必须的元素,根瘤菌结瘤因子的硫化修饰与其宿主范围和识别效率有着重要关系,实验室前期研究中突变费氏中华根瘤菌的cys DN基因后,突变体不能利用硫酸盐生长,这与Sinorhizobium sp.BR816的cys D基因突变后的现象不同,两株菌的硫酸盐同化途径中可能存在差异.为探讨不同根瘤菌cys DN基因对硫酸盐同化途径的影响,本研究利用同源交换的方法构建费氏中华根瘤菌WGF03、费氏中华根瘤菌HN01、苜蓿中华根瘤菌14500及大豆慢生根瘤菌15606的cys DN突变体,Δcys DN-WGF03、Δcys DN-HN01、Δcys DN-14500和cys NR-15606,对突变体的硫源利用情况和植株结瘤情况进行研究.结果发现,Δcys DN-WGF03和Δcys DN-HN01失去利用硫酸盐的能力;Δcys DN-14500能微弱地利用硫酸盐生长,生长量约为野生菌株的1/3左右;cys NR-15606对硫酸盐的利用与野生菌株相比无明显差别.植株实验结果显示,Δcys DN-WGF03、Δcys DN-HN01和Δcys DN-14500在平均瘤数、平均瘤重、平均植株干重和固氮酶活性方面均表现出显著降低,而突变体的回补体能够恢复硫酸盐的利用能力及与植株的共生固氮能力.这说明cys DN基因在费氏中华根瘤菌WGF03、费氏中华根瘤菌HN01和苜蓿中华根瘤菌14500硫酸盐同化途径中起着关键影响,而该基因在大豆慢生根瘤菌15606的影响并不明显.本研究表明,cys DN基因在不同根瘤菌中所起的作用不同,不同根瘤菌的硫酸盐同化方式也存在差异.     

氨氮胁迫下菲律宾蛤仔肝胰腺内参基因的筛选

利用荧光定量PCR技术对目标基因进行转录水平分析,选择合适的内参基因作为对照是其前提条件.随着氨氮胁迫菲律宾蛤仔毒性效应的深入研究,需要准确评价氨氮对蛤仔肝胰腺组织基因表达的影响.然而,不同浓度氨氮条件下的菲律宾蛤仔肝胰腺组织的内参基因的筛选尚未报道.本研究以2种不同氨氮浓度暴露菲律宾蛤仔14 d的肝胰腺组织cDNA为qRT-PCR的模板,利用geNorm、NormFinder、BestKeeper和RefFinder软件评估β-肌动蛋白(Actin)、延伸因子1-α(EF-1α)、β-微管蛋白(Tubu)、18S核糖体RNA(18S)、泛素(Ubi)和亲环蛋白A(CyPA)6个内参基因的表达稳定性.结果表明,肝胰腺组织中备选内参基因的表达稳定性依次为:Actin>18S>CyPA>Ubi>Tubu>EF-1α,即Actin为表达最稳定的内参基因.本研究为进一步分析氨氮胁迫下菲律宾蛤仔肝胰腺组织的基因表达情况,提供了可靠的内参基因.     

Effects of protein-constrained brood food on honey bee (<Emphasis Type="Italic">Apis mellifera</Emphasis> L.) pollen foraging and colony growth

Pollen is the sole source of protein for honey bees, most importantly used to rear young. Honey bees are adept at regulating pollen stores in the colonies based on the needs of the colony. Mechanisms for regulation of pollen foraging in honey bee are complex and remain controversial. In this study, we used a novel approach to test the two competing hypothesis of pollen foraging regulation. We manipulated nurse bee biosynthesis of brood food using a protease inhibitor that interferes with midgut protein digestion, significantly decreasing the amount of protein extractable from hypopharyngeal glands. Experimental colonies were given equal amounts of protease inhibitor-treated and untreated pollen. Colonies receiving protease inhibitor treatment had significantly lower hypopharyngeal gland protein content than controls. There was no significant difference in the ratio of pollen to nonpollen foragers between the treatments. Pollen load weights were also not significantly different between treatments. Our results supported the pollen foraging effort predictions generated from the direct independent effects of pollen on the regulation of pollen foraging and did not support the prediction that nurse bees regulate pollen foraging through amount of hypopharyngeal gland protein biosynthesis.     

Biotic and abiotic stress effects on nitrogen chemistry in the salt marsh cordgrassSpartina alterniflora (Poaceae)

Summary Planthopper (Insecta: Homoptera) feeding stress induces a senesence-like response in the leaves ofSpartina alterniflora characterized by decreased soluble protein, an increased total amino acid pool, and elevated levels of 10 individual amino acids. Increased proline and tryptophan in response to planthopper feeding could not be fully explained by protein degradation. Low degrees of soil salinity stress resulted in an increased total free amino acid pool and elevated levels of 7 amino acids. Anaerobic soil stress resulted in decreased glutamic acid and increased asparagine. Low salinity and anaerobic stress had no effect on soluble protein levels. Glycinebetaine was not affected by the stresses examined in this study.     

Humic Acids from Endemic Arsenicosis Areas in Inner Mongolia and from the Blackfoot-Disease Areas in Taiwan: A Comparative Study

The physical and chemical properties of humic acids (HA) extracted from drinking waters from the endemic arsenicosis areas in Inner Mongolia and from the Blackfoot disease (BD) areas in Taiwan are studied by using AAS (atomic absorption spectrophotometry), ICP-MS (inductively coupled plasma emission-mass spectrometry), IR (Infrared spectroscopy), FR (fluorescence spectrometry) and TLS (total luminescence spectroscopy) in order to shed light on the pathogenesis of BD with the concern as to whether the disease may eventually occur in arsenicosis-affected areas in the Mainland of China. Ames test and lipid peroxidation experiments were also conducted on these HA samples. It is found that water samples from the two regions are high in arsenic (As) with strong fluorescence and apparent positive correlations between As content, fluorescence, pH and total dissolved solids (TDS). The water samples are similar in fluorescence spectra but differ somewhat in IR and TLS between the two regions. The difference may be a reflection of the difference in radicals and structure of the HA owing to different hydrogeological conditions, and may also be related to the difference in their biological effects, i.e., HA from Inner Mongolia have a stronger ability to cause lipid peroxidation while HA from Taiwan exhibits a more prominent effect of mutation with respect to TA98(±S9).     

威海荣成鹿角菜野生种群遗传多样性及资源保护策略

利用随机扩增多态性DNA（RAPD）方法对威海荣成地区鹿角菜（Silvetiasiliquosa）野生种群共30个个体的遗传多样性水平及遗传结构进行研究。结果表明,利用21条随机引物共检测到112个多态性位点,多态性位点百分率为93．75％,Nei基因多样性指数为0．3515±0．1352,Shannon多样性指数为0．5046±0．1126。通过聚类分析而划为一类的3个亚种群间的遗传分化指数为0．1349～0．2189,基因流为1．7793。研究结果表明威海荣成地区的鹿角菜种群遗传多样性较高,种群内存在较为明显的遗传分化,种群遗传资源不受遗传漂变的影响。针对当地鹿角菜遗传资源的现状,应加强现存种群的就地保护,恢复种群规模;进行取样养殖保护时则需避免因近交而造成种群资源退化。     

The influences of different organic fractions in refuse on the sorption and bioavailability of dibutyl phthalate

The structure and composition of the organic matter in landfilled refuse might have an influence on the migration and transformation of dibutyl phthalate (DBP). Humic acid (HA) and humin (HU) were separated from aged-refuse to determine the influences of different organic fractions in the refuse on the sorption and bioavailability of DBP. The sorption kinetics and isotherms for the sorption of DBP to HA, HU, and whole refuse were determined. The results showed that the sorption constants (K) and nonlinearities decreased in the order HU?>?whole refuse?>?HA. The HA had lower K values than did the other refuse fractions, and it retarded the biodegradation of DBP over a short degradation period (48?h). Increasing the amount of HA present caused the amount of DBP that was biodegraded to decrease significantly, 81.3% of the DBP sorbed to HA being degraded in the original experiments after 48?h but 21.8% of the DBP being degraded when three times as much HA was used. Similar results were not observed when the amount of HU used was changed. These findings suggest that HA plays an important role in the biodegradation of DBP adsorbed by refuse.     

热休克、Poly I:C上调草鱼GRP78基因表达

葡萄糖调节蛋白78(GRP78)是细胞内一种应急蛋白,它通过对细胞内、外胁迫因子产生强烈的应答而调节内质网稳态.草鱼(Ctenopharyngodon idellus)GRP78克隆于草鱼肝肾cDNA文库,全长2 490 bp.其中,5'非编码区为155 bp,含有7个热休克元件,3'非编码区为373 bp,最大开放阅读框为1 962 bp,编码653个氨基酸.序列分析表明,草鱼GRP78的N端包含3个HSP70家族的签名序列,C末端为内质网蛋白滞留信号KDEL,草鱼GRP78与人及其它动物的同源性很高.适应性进化分析也显示GRP78非常保守,受到强烈的功能约束,说明该蛋白质在进化中非常重要.RT-PCR分析表明,相对于本底水平,34℃热激和Poly I:c诱导后,草鱼GRP78在肝、肾等组织巾的表达明显上调.图4表1参16     

Population regulation by enemies of the grass Brachypodium sylvaticum: demography in native and invaded ranges

The enemy-release hypothesis (ERH) states that species become more successful in their introduced range than in their native range because they leave behind natural enemies in their native range and are thus "released" from enemy pressures in their introduced range. The ERH is popularly cited to explain the invasive properties of many species and is the underpinning of biological control. We tested the prediction that plant populations are more strongly regulated by natural enemies (herbivores and pathogens) in their native range than in their introduced range with enemy-removal experiments using pesticides. These experiments were replicated at multiple sites in both the native and invaded ranges of the grass Brachypodium sylvaticum. In support of the ERH, enemies consistently regulated populations in the native range. There were more tillers and more seeds produced in treated vs. untreated plots in the native range, and few seedlings survived in the native range. Contrary to the ERH, total measured leaf damage was similar in both ranges, though the enemies that caused it differed. There was more damage by generalist mollusks and pathogens in the native range, and more damage by generalist insect herbivores in the invaded range. Demographic analysis showed that population growth rates were lower in the native range than in the invaded range, and that sexually produced seedlings constituted a smaller fraction of the total in the native range. Our removal experiment showed that enemies regulate plant populations in their native range and suggest that generalist enemies, not just specialists, are important for population regulation.