烟蚜热激蛋白Hsp90基因的克隆及在UV-B胁迫下的表达分析

为探讨UV-B胁迫对烟蚜Myzus persicae热激蛋白Hsp90基因表达量的影响,采用RT-PCR与RACE技术克隆了烟蚜热激蛋白Hsp90基因的全长,并对其进行生物信息学分析,利用实时荧光定量PCR技术研究了烟蚜Hsp90基因在不同时长UV-B胁迫下的表达量变化。结果表明,烟蚜Hsp90基因的cDNA全长为2 670 bp,编码728个氨基酸,编码蛋白质的相对分子量为82.6 kD,等电点为4.95,获得的氨基酸序列具有Hsp90蛋白家族的1个签名序列及C末端MEEVD基序,推测其属于胞质型热激蛋白。系统进化树结果显示,烟蚜Hsp90与其它昆虫Hsp90具有很高的相似性。实时荧光定量PCR结果表明,不同时长UV-B胁迫下烟蚜Hsp90均有表达,随着照射时间延长,Hsp90表达量表现为先上升后下降的趋势;与对照相比,照射时间为15、30、60、90和120 min时,Hsp90表达量均显著升高,且在60 min时Hsp90表达量达最大,是对照组的2.05倍。表明Hsp90基因在不同时长UV-B胁迫下差异表达,在烟蚜适应紫外胁迫的分子机制中具有重要作用。     

水稻二化螟热激蛋白的研究进展

水稻二化螟Chilo suppressalis(Walker)是水稻上的主要害虫之一,近年来在部分地区发生为害逐渐加重。热激蛋白是一类胁迫蛋白,在细胞生长、发育、分化、基因转录等功能方面具有重要的作用。本文综述了二化螟热激蛋白的研究进展。目前,已在二化螟体内鉴定出11种热激蛋白,它们分别属于热激蛋白90家族、热激蛋白70家族、热激蛋白60家族和小分子量热激蛋白家族。这些热激蛋白在二化螟生长发育中的调节作用也各不相同;同时,热激蛋白(Cshsp90,Cshsp70和Cshsp60)还与水稻二化螟的滞育有着紧密的联系。不同的热激蛋白对温度的响应大体可以分为三种类型:对高低温都有响应型、对低温响应型和对温度不响应型。最后,分析了二化螟热激蛋白研究过程中存在的问题,提出了该研究领域的展望。     

Plant invasiveness and target plant density: high densities of native Schima wallichii seedlings reduce negative effects of invasive Ageratina adenophora

Economically feasible strategies to cope with invasive species are urgently needed. Plant density can be increased to reduce competitive effects on target plants. This study indicates that increasing native plant density can be used to reduce the effect of invasive Ageratina adenophora. Seedlings of an indigenous tree species, Schima wallichii, were grown in pots containing uninvaded or invaded soil, with or without A. adenophora leaf litter on the soil surface. Schima wallichii seedlings were also grown at four densities under four levels of A. adenophora leaf litter. Root and shoot biomass and length were measured as response parameters in both bioassays. Schima wallichii growth was inhibited by A. adenophora leaf litter and invaded soil. High litter levels reduced S. wallichii root length and dry weight at low plant densities. The inhibition disappeared at high S. wallichii plant densities. As A. adenophora did not inhibit S. wallichii growth at high plant densities, adjustments of seedling density should be studied as a possible management strategy for invasion by A. adenophora and potentially by other exotic plant species. As density‐dependent growth inhibition is the key characteristic of chemical interference, we propose that phytotoxins contribute to A. adenophora invasion particularly at low densities of native seedlings.     

Cloning,characterisation and expression profiling of the cDNA encoding the ryanodine receptor in diamondback moth,Plutella xylostella (L.) (Lepidoptera: Plutellidae)

BACKGROUND: The rynodine receptors (RyRs) are the main targets of diamide insecticides such as chlorantraniliprole. To provide the basis for a good understanding of the molecular mechanisms of diamide insecticide resistance, an RyR gene from Plutella xylostella was cloned and characterised in the present paper. RESULTS: A full‐length cDNA sequence of RyR was cloned from P. xylostella through RT‐PCR and rapid amplification of cDNA ends (RACE). The gene (named PxRyR1) is 15 753 bp long, with an open reading frame of 15 354 bp, encoding a predicted RyR of 5117 amino acids. An alternative splicing of the PxRyR1 was also cloned and named PxRyR2. The PxRyR1 shares 77–93% identity with other insect RyRs. Quantitative real‐time PCR analysis showed that the PxRyR was expressed at a high level in second‐instar larvae and adults, at a low level in prepupae and pupae and abundantly in the body wall muscle and head (respectively 6.00 and 3.12 times the expression in the gut). Western blot analysis with anti‐RyR antibodies showed that the RyR was mainly present in the body wall muscle and head, but barely present in the haemocyte and gut. CONCLUSIONS: There are at least two alternative splices of PxRyR expressed in all developmental stages and tissues in P. xylostella at various levels. The results provided the basis for further understanding of the mechanisms of resistance to diamide insecticides in P. xylostella. Copyright © 2012 Society of Chemical Industry     

松材线虫Hsp70基因的克隆与原核表达

 热激蛋白70(Hsp70)是已知热休克蛋白家族中最重要的-种, 它在细胞内的大量表达可以明显改善细胞的生存能力, 提高对环境胁迫或伤害的耐受性。采用RACE-PCR技术, 从松材线虫(Bursaphelenchus xylophilus)中克隆了Hsp70基因的全长cDNA序列(共2 061 bp)(GenBank登录号为:DQ785812)。其编码-个分子量为70 kD的642个氨基酸的蛋白序列, 含有3段Hsp70家族的签名序列。同源性分析表明, 氨基酸序列与其它真核生物的Hsp70序列具有很高的相似性, 并与秀丽隐杆线虫(Caenorhabditis elegans)热激蛋白70家族中的hsp-1基因编码的氨基酸序列更为相似。因此, 将克隆的松材线虫Hsp70基因命名为Bx-hsp-1。构建了-个原核表达载体Bx70pEASY-E1, 当IPTG终浓度为0.4~0.8 mmol/L时, 能诱导表达融合蛋白。Bx-hsp-1基因的克隆和表达, 将为松材线虫的生态适应性机理研究奠定基础。     

Increased free amino acid contents of the invasive weed Ageratina adenophora and the effects on its specialist herbivore Procecidochares utilis

In order to verify the escape‐from‐enemy hypothesis from the changes of nutrient substance and fitness of natural enemies on alien plants, contents of free amino acids in native and invasive plant populations of Ageratina adenophora and life history parameters of specialist herbivore Procecidochares utilis reared on these plants were investigated. Our results showed that the contents of glycine, valine, γ‐aminobutyric acid, proline, serine, alanine, and arginine in the invasive plants were higher than those in the native plants of A. adenophora. There was a shorter developmental duration and higher fecundity of P. utilis when fed on the invasive plants. The results indicated a possible fitness tradeoff of natural enemies between invasive and native plants arose from nutrient substance changes.     

紫外线、高压静电和大麦黄矮病毒胁迫下荻草谷网蚜SOD基因表达

为探索环境胁迫影响昆虫适合度的内在机制,通过cDNA末端快速扩增（rapid amplification of c DNA end,RACE）技术克隆得到荻草谷网蚜Sitobion miscanthi体内超氧化物歧化酶（superoxide dismutase,SOD）基因MnSOD和CuZnSOD的cDNA序列全长,对该序列进行分析,检测紫外线、高压静电和大麦黄矮病毒（barley yellow dwarf virus,BYDV）胁迫下荻草谷网蚜体内MnSOD和CuZnSOD基因表达量的变化。结果显示,荻草谷网蚜体内MnSOD和CuZnSOD基因的cDNA序列全长分别为1 517 bp和954 bp(GenBank登录号分别为MT533627和MT533626),开放阅读框分别为669 bp和459 bp,分别编码222个和152个氨基酸,预测蛋白质相对分子量分别为24.99 kD和15.84 kD。荻草谷网蚜体内MnSOD和CuZnSOD蛋白均与半翅目蚜科昆虫同源蛋白氨基酸序列相似性高,亲缘关系最近。0.50 m W/cm2（低强度）和0.70 m W/cm2（高强度）紫外线胁迫下...     

向日葵3个谷胱甘肽-S-转移酶GST基因的克隆及表达模式分析

从向日葵转录组测序结果中获得了3个对盐胁迫有响应的谷胱甘肽-S-转移酶（GST,EC2.5.1.18）GST基因,构建了系统进化树并进行分析,得知这3个基因属于Tau型GST,并将它们命名为HaGSTU26（HanXRQChr04g0127901）、HaGSTU8（HanXRQChr06g0177581）、HaGSTU27（HanXRQChr10g0316331）,然后以向日葵Sk02R为试验材料,克隆这3个基因,并进行了不同组织和不同胁迫条件下的表达分析。序列分析表明：HaGSTU26基因组为1674bp,CDS（编码蛋白序列）长666bp,编码221个氨基酸,由2个外显子和1个内含子组成;HaGSTU8基因组为2271bp,CDS长654bp,编码217个氨基酸,由2个外显子和1个内含子组成; HaGSTU27基因组为663bp,CDS长663bp,编码220个氨基酸,由1个外显子组成。实时荧光定量PCR分析表明：向日葵HaGSTU26、HaGSTU8、HaGSTU27基因在不同组织（根、幼叶、成熟叶、茎、幼茎、苞叶）中表达量不同,其中,HaGSTU26基因和HaGSTU27基因在根中表达量最高,而HaGSTU8基因在苞叶中表达量最高,但这3个基因均在成熟茎中的表达量最低。在不同胁迫条件下,测定这3个基因在向日葵幼苗中的表达量,结果表明在盐及ABA胁迫下,基因表达量均随着处理时间的增加而呈现先增加后下降的趋势。其中,在盐胁迫下,HaGSTU26基因在12 h后相对表达量最高,HaGSTU8及HaGSTU27基因表达量在3h后相对表达量最高。在ABA胁迫后,HaGSTU26及HaGSTU27基因表达量在12 h后相对表达量最高,HaGSTU8在24 h后相对表达量最高。在冷胁迫下,HaGSTU26和HaGSTU27基因上调表达,它们分别在3、24 h后相对表达量最高,HaGSTU8基因下调表达,其相对表达量随处理时间的延长呈现逐渐减少的趋势。在热胁迫条件下,这3个基因的相对表达量随着胁迫时间的延长而增加,均在24 h后表达量最高。以上结果说明这3个基因对不同非生物胁迫（盐、ABA、冷、热胁迫）均有响应。     

Molecular cloning and characterization of the cinnamate 4‐hydroxylase gene from Eupatorium adenophorum

Cinnamate‐4‐hydroxylase (CA4H), a cytochrome P450‐dependent monooxygenase, plays crucial roles in phenylpropanoid metabolism and plant defense. Previously, the authors showed that the expression of CA4H was induced in response to an allelopathic substance in Eupatorium adenophorum. Here, the full‐length cDNA of EaCA4H was cloned by using rapid amplification of cDNA ends. The 1518 bp open reading frame of EaCA4H was deduced to encode a protein of 505 amino acid residues. Like other CA4H proteins, the predicted EaCA4H polypeptides contained conserved domains of cytochrome P450. A Southern blot analysis indicated that at least five copies of EaCA4H exists in the genome of E. adenophorum. Subcellular localization revealed nuclear‐localized EaCA4H–green fluorescent protein fusion protein in onion epidermal cells. Heterologous silencing of endogenous CA4H in tobacco by a conserved EaCA4H fragment resulted in reduced expressions of key enzymatic genes and the production of downstream flavonoids in the phenylpropanoid pathway. Intriguingly, similar effects were observed in transgenic tobacco plants overexpressing EaCA4H. Altogether, the results indicate that the disturbed expression of CA4H in plants leads to relatively low expression levels of key enzymatic genes and the accumulation of the flavonoids that are involved in phenylpropanoid metabolism.     

粘虫脂动激素基因MsepAKH1的克隆与成虫期表达模式分析

为明确脂动激素基因AKH在粘虫Mythimna separata(Walker)能源代谢过程中的作用,采用RT-PCR和RACE技术克隆得到粘虫脂动激素基因MsepAKH1的cDNA全长,并通过实时荧光定量PCR技术测定该基因在成虫期的组织与发育阶段表达模式。结果表明,粘虫脂动激素基因MsepAKH1的cDNA序列全长为449 bp,GenBank登录号为KP979739.1,开放阅读框为207 bp,编码68个氨基酸,具有昆虫脂动激素基因家族典型的结构特征;预测该基因编码的MsepAKH1蛋白分子量为7.61 kD,等电点为8.46,MsepAKH1的氨基酸序列与烟草天蛾Manduca sexta、二化螟Chilo suppressalis、小菜蛾Plutella xylostella、棉铃虫Helicoverpa armigera、家蚕Bombyx mori的AKH氨基酸序列同源性较高。MsepAKH1基因在粘虫初羽化雌蛾不同组织间的表达量差异显著,主要在头部和飞行肌内表达,分别约为同日龄雌蛾脂肪体内表达量的15.4倍与8.1倍,在脂肪体、卵巢与中肠内的表达量显著降低。对不同日龄雌蛾头部和飞行肌内的表达量检测发现,在7日龄雌蛾头部和3日龄雌蛾飞行肌内的表达量分别显著高于其它日龄雌蛾相同组织中的表达量,分别约为初羽化雌蛾相同组织表达量的2.4倍与1.6倍。表明MsepAKH1基因的表达因粘虫雌蛾组织与日龄间的不同而呈现动态变化。     

Hsp70 and Hsp90 expression in citrus and pepper plants in response to <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">citri</Emphasis>

Hsp70 and Hsp90 expression in response to high and low temperatures was studied in orange, the host plant of Xanthomonas axonopodis pv. citri and in a non-host resistant plant, pepper. As expected in both plants, the expression of these chaperones was induced at high temperatures while at cold temperatures the response was chaperone and plant-dependent. Expression of Hsp70 and Hsp90 was analysed during citrus canker and no changes in their levels could be observed whereas pepper plants infiltrated with the phytopathogen showed an increase in the levels of both chaperones. These results suggest that no changes in Hsp70 and Hsp90 expression are necessary during the disease while they are increased in non-host resistance.     

小麦白粉菌HSP70基因的克隆及高温胁迫对其表达的影响

小麦白粉病是由专性寄生菌Blumeria graminisf.sp.tritici引起的一种重要的气传真菌病害,该病曾于1990-1991年两年在我国发生大流行,近年来其发生面积也一直维持在500万公顷以上,给我国小麦生产造成了严重的损失.已有的研究结果表明,温度不但影响小麦白粉病的发生和流行,而且温度胁迫作用还会对白粉病菌群体产生影响,从而使病菌群体对温度的敏感性降低.目前已发现在我国白粉病菌群体中存在抗高温或耐高温的菌株,而且这些菌株在较高的温度下其寄生适合度要高于对温度高敏感性的菌株[1].由于病菌群体对温度敏感性的变化有可能影响病害越夏和越冬的范围,进而影响病害的发生和流行程度,因此,有必要对其耐高温的分子机制进行研究,这对于了解小麦白粉菌对温度的适应性以及气候变暖对此病害发生和流行的长期影响具有重要的意义.     

Comparison of induced resistance activated by benzothiadiazole, (2R,3R)‐butanediol and an isoparaffin mixture against anthracnose of Nicotiana benthamiana

Resistance in Nicotiana benthamiana against anthracnose caused by the hemibiotrophic fungus Colletotrichum orbiculare was activated by benzothiadiazole (BTH), (2R,3R)­butanediol or PC1, an isoparaffin‐based mixture. In inoculation experiments, BTH, (2R,3R)­butanediol and PC1 reduced the number of lesions per leaf area caused by C. orbiculare by 98%, 77% and 81%, respectively. Foliar application of BTH induced expression of genes for the acidic pathogenesis‐related (PR) proteins, NbPR‐1a, NbPR‐3Q and acidic NbPR‐5. In contrast, soil application of (2R,3R)­butanediol or PC1 primed expression of genes for the basic PR proteins, NbPRb‐1b, basic NbPR‐2 and NbPR‐5dB. These results are consistent with the activation of salicylic‐acid‐dependent systemic acquired resistance (SAR) by BTH and that of jasmonate/ethylene‐dependent induced systemic resistance (ISR) by (2R,3R)­butanediol or PC1, and show that (2R,3R)­butanediol and PC1 can affect gene expression similarly to plant growth‐promoting rhizobacteria. However, the effects of (2R,3R)‐butanediol and PC1 were not identical. In addition to priming, (2R,3R)‐butanediol induced expression of basic NbPR‐2, whereas PC1 treatment induced expression of both NbPRb‐1b and basic NbPR‐2. Although a number of microbial products, such as (2R,3R)­butanediol, have been shown to produce ISR, this is the first demonstration that an isoparaffin‐based mixture, not derived from a microorganism, can produce ISR.     

Biology and management of the invasive weed Ageratina adenophora (Asteraceae): current state of knowledge and future research needs

Biological invasion is increasing worldwide and the management of invasive species is becoming an important priority for vegetation managers. Success of invasive species management depends on a thorough understanding of the biology of the organism in question and the effectiveness of current management efforts, in order to identify the best practices for management improvement. In this review, we synthesised current biological knowledge of a noxious invasive weed Ageratina adenophora to identify knowledge gaps and assessed management efforts to identify best practices. Finally, we proposed some priority areas for future research to fill knowledge gaps and improve management. Our analysis showed that A. adenophora has already invaded 40 countries, mainly in Asia, Oceania, Africa and Europe. Phenotypic plasticity, allelopathic interference and invasion‐mediated changes in the soil microbial community are the proposed mechanisms that facilitate rapid spread of this weed. However, allelopathy as a mechanism of invasion success of this weed has not been supported by ecologically meaningful experiments. Though mechanical, chemical and biological control measures have been used, their success remains limited and the weed continues to spread in new regions. Among seven biological control agents examined to date, gall fly (Procecidochares utilis) and leaf spot fungus (Passalora ageratinae) have been effective in limited areas to suppress growth of this weed. Some perennial native grasses (e.g. Setaria sphacellata and Lolium perenne) have shown potential to competitively suppress A. adenophora. In conclusion, understanding the invasion mechanisms, exploring further to identify effective biological control agents, combined with approaches of ecological restoration, could help in the management of this weed.     

姜黄素对朱砂叶螨几丁质酶基因表达的影响

为明确植物源活性物质姜黄素对朱砂叶螨Tetranychus cinnabarinus的杀螨作用机理,克隆并分析了6条朱砂叶螨几丁质酶基因(TcCHTs)的cDNA序列,利用RT-qPCR技术,测定了不同发育阶段的螨体内几丁质酶基因表达的情况,同时研究了姜黄素作用下几丁质酶基因表达的变化。结果表明:6条几丁质酶基因(TcCHIT1、TcCHIT2、TcCHIT3、TcCHT4、TcCHT5和TcCHT6)有不同的结构域,预测其蛋白质分子质量分别为60.80、69.03、104.32、59.75、45.63和49.05 kDa,等电点分别为5.44、8.65、6.23、6.07、6.12和8.27。6条基因的表达量均为幼螨和若螨时期显著高于卵和成螨时期。不同浓度姜黄素处理朱砂叶螨若螨24 h后,6条基因的表达量均有不同程度下调,其中TcCHIT1和TcCHT5的下调更为显著。研究结果表明,姜黄素对朱砂叶螨的杀螨作用机制之一可能是通过抑制几丁质酶的活性,进而影响几丁质的降解过程,最终因螨体壁受损而导致螨不能正常生长发育。     

舞毒蛾DnaJ1基因克隆分析及对甲萘威胁迫响应

Dna J蛋白是Dna K/Hsp70的辅助分子伴侣,通过调节Hsp70的ATPase活性来影响蛋白复合体的合成与组装。为明确舞毒蛾Lymantria dispar的LdDnaJ1基因特性及对杀虫剂甲萘威的胁迫响应,通过克隆LdDnaJ1全长基因并运用实时荧光定量RT-PCR技术测定了甲萘威对其LdDnaJ1基因表达量的影响。结果表明,舞毒蛾Ld Dna J1全长基因开放阅读框为1 062 bp,编码353个氨基酸,分子质量为39.91 kD,理论等电点为5.65;舞毒蛾Dna J与柑橘凤蝶Papilio xuthus Dna J亲缘关系较近。甲萘威对舞毒蛾2龄幼虫24 h和48 h的致死中浓度LC50分别为74.04 mg/L和31.48mg/L。低剂量(LC_5、LC_(10)和LC_(30))甲萘威胁迫下,舞毒蛾2龄幼虫Ld Dna J1基因表达量均下调,LC_(30)甲萘威处理后72 h时LdDnaJ1基因表达量最低,仅为对照的15.70%。表明甲萘威可抑制舞毒蛾LdDnaJ1基因的表达,且呈现明显的时间和剂量效应。     

泽兰实蝇寄生对紫茎泽兰生长和防御能力的影响

为明确泽兰实蝇Procecidochares utilis寄生对紫茎泽兰Ageratina adenophora生长及防御能力的影响,在温室内设置泽兰实蝇寄生与未寄生紫茎泽兰2组盆栽处理,测定泽兰实蝇寄生对紫茎泽兰生长特性、生物量、叶片营养物质、次生代谢物质和保护酶的影响。结果表明,泽兰实蝇寄生显著降低了紫茎泽兰的株高、茎粗、叶片厚度和叶片数,分别显著降低了24.80%、16.86%、42.31%和27.82%;同时,地上生物量、根部生物量和总生物量分别显著降低了58.89%、54.29%和57.60%。泽兰实蝇寄生还导致紫茎泽兰的营养成分发生显著变化,其中可溶性蛋白、可溶性糖、可溶性淀粉和叶绿素含量分别显著降低了41.18%、11.75%、34.57%和29.96%。泽兰实蝇寄生改变了紫茎泽兰的保护酶活性,其过氧化氢酶和超氧化物歧化酶活性分别比未寄生植株显著升高了59.36%和108.06%,过氧化物酶活性降低了25.69%;同时使紫茎泽兰单宁酸含量显著降低了21.67%,总酚和类黄酮的含量分别显著上升了1.21倍和2.09倍。泽兰实蝇寄生的紫茎泽兰总生物量与次生代谢物质总酚和类黄酮的含量呈显著负相关,相关系数r分别为-0.06和-0.43,对照组则不存在相关性。表明泽兰实蝇寄生可能促使紫茎泽兰改变自身的生长与防御能量分配,减少了营养和生长的投入,将更多的资源投向繁殖和防御。