巴西橡胶树线粒体50S核糖体蛋白L21cDNA的克隆与分析

在橡胶生产中,死皮生理综合症严重制约了橡胶树(Hevea brasiliensis)单产的提高。在早期构建的差减文库中,筛选到一条在死皮植株中下调表达的基因片段,该片段编码的蛋白与线粒体50S核糖体蛋白L21(mRPL21)同源。通过ESTs序列拼接和RT-PCR,获得一条853 bp的cDNA序列(命名为HbmRPL21,GenBank登录号为HM800425),该序列包含一个完整的开放读码框,编码271个氨基酸,理论分子量为30.52 kD,等电点为8.40。同源比对表明,植物和动物界间mRPL21序列差异很大,而植物界内则相对比较保守。生物信息学分析表明,HbmRPL21是一个包含Ribosomal_L21p保守结构域的线粒体定位蛋白。     

Hop Latent Viroid (HLVd) Microevolution: An Experimental Transmission of HLVd “thermomutants” to Solanaceous Species

The possibility was examined whether the pool of sequence variants of HLVd which accumulated as progeny of “thermomutants” induced upon heat-treatment of hop could initiate infection of non-host solanaceous plants. It was found that HLVd microevolution led to the appearance of HLVd population in tomato. This viroid population was maintained at levels detectable by molecular hybridisation, showing the highest concentration in apical leaves. HLVd was further transferred from tomato to Nicotiana benthamiana, where distinct HLVd sequence variants appeared and were stably maintained at low levels. Our results show that replication of HLVd under heat stress resulted in the production of viroid quasispecies, potentially important for viroid evolution in so-called non host plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Continous replication of potato spindle tuber viroid (PSTV) in permanent cell cultures of potato and tomato

The continuous replication of potato spindle tuber viroid (PSTV) in callus cultures from PSTV-infected wild-type potato (Solanum dem/ssum L.) and tomato (Lycopersicon peruvianum L. Mill) plants and in cell suspensions derived from potato protoplasts (Solanum tuberosum L.) inoculatedin vitro is described. The persistence of PSTV replication in these cell lines through at least 14 subculture passages, which corresponds to a continous replication over a period of more than one year, was demonstrated by infectivity assay and by polyacrylamide-gel electrophoresis of isolated nucleic acids. This continuous synthesis denovo of PSTV was substantiated by the incorporation of [³H]uridine and of [³²P]orthophosphate into viroid RNA.     

Occurrence of Mosaic Mottle, a Viroid Disease of Pigeonpea (Cajanus cajan) in India

During investigations of viroid diseases of food crops in Delhi, India, an infectious low-molecular-weight RNA (M_r 1.3 × 10⁵) was isolated from pigeonpea (Cajanus cajan) plants showing mosaic mottling and reduced size of leaves, stunting and no flowering. This RNA infected mechanically inoculated C. cajan, Chenopodium amaranticolor, Nicotiana glutinosa, and N. clevelandii. The causal agent, for which the name pigeonpea mosaic mottle viroid (PMMVd) is proposed, differed from N. glutinosa stunt viroid (NgSVd), the only viroid reported to infect legumes, in size and symptom expression on the original host species.     

死皮对橡胶树树皮线粒体超微结构及活性氧代谢的影响

为了解析橡胶树（Hevea brasiliensis）死皮的发生机制,有效进行死皮防治,以健康、轻度死皮、重度死皮橡胶树树皮为材料,研究死皮发生过程中树皮线粒体超微结构变化规律及活性氧（ROS）相关基因的表达模式变化。结果表明：死皮树线粒体超微结构发生不规则形变,膜内基质溶解,嵴消失,内腔空泡化等,且严重程度与死皮严重程度成正比。荧光定量PCR结果表明,过氧化物酶基因HbPOD2和HbPOD3在死皮树中的表达量高于健康树,可作为监测割胶强度、刺激强度和死皮发生的“标志”基因。植物细胞重要ROS清除酶过氧化氢酶基因HbCAT在死皮树中也下调表达,预示ROS产生与清除之间的平衡是影响橡胶树死皮发生的关键因素。橡胶树中重要抗氧化代谢物基因表达结果表明,HbGST1、HbGST2和HbPPO在死皮树中的表达量均高于健康树,可能与死皮发生过程胶乳原位凝固相关。本研究通过揭示死皮发生过程树皮超微结构和ROS相关基因表达模式变化,为阐明橡胶树死皮发生机制提供新观点,同时为进一步开发监测割胶强度、刺激强度和死皮发生的基因“标志”提供理论基础。     

RNA-dependent RNA polymerase 1 delays the accumulation of viroids in infected plants

RNA-dependent RNA polymerase 1 (RDR1) is essential for plant antiviral defence, but its role in plant defence against viroid infection remains unknown. The present study aimed to identify the function and mechanism of RDR1 in plant resistance to viroid infection. Overexpression of Nicotiana tabacum RDR1 (NtRDR1) delayed the accumulation of potato spindle tuber viroid (PSTVd) genomic RNA and PSTVd-derived small RNA (sRNA) in Nicotiana benthamiana plants at the early invasion stage, but not in the late stage of infection. Conversely, virus-induced gene silencing of tomato RDR1 (SlRDR1a) increased the susceptibility to PSTVd infection (increased viroid accumulation). Salicylic acid (SA) pretreatment induced SlRDR1a expression and enhanced the defence against PSTVd infection in tomato plants. Our study demonstrated that RDR1 is involved in SA-mediated defence and restricts the early systemic invasion by PSTVd in plants. The decreased PSTVd accumulation in N. benthamiana was not caused by efficient accumulation of PSTVd sRNAs. These results deepen our understanding of the mechanism of RDR1 in plant defence responses to viroid attack.     

Cloned single- and double-stranded DNA copies of potato spindle tuber viroid (PSTV) RNA and co-inoculated subgenomic DNA fragments are infectious

A set of monomeric and oligomeric potato spindle tuber viroid (PSTV) specific DNA forms representing complete DNA copies of the circular PSTV RNA genome were constructed and cloned in plasmid pBR322 and bacteriophage M13. Both single- and double-stranded PSTV DNAs are capable of initiating viroid replication in mechanically inoculated tomato plants where it normally proceeds via the RNA-RNA pathway without DNA being involved. All dimeric and higher multimeric forms were infectious irrespective of their polarity in the case of single-stranded DNA and regardless of their orientation in the vector DNA in the case of double-stranded DNA. The vector-inserted monomeric PSTV DNA units were also found to be infectious but of low specific infectivity which was increased when these monomers had been excised. Even two subgenomic DNA fragments, representing together the 359 nucleotides of the PSTV RNA genome, initiated the synthesis of viroid RNA progeny when co-inoculated although each fragment by itself is non-infectious. These results are discussed with respect to the infectivity previously observed with certain cloned DNAs of conventional RNA and DNA viruses.     

Ethanol treatment triggers a heat shock-like response but no thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings

Non‐lethal heat‐shock (HS) treatment has previously been shown to induce thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings. This acquired thermotolerance correlates with the de novo synthesis of heat‐shock proteins (HSPs). Interestingly, we found that ethanol treatments also elicited HS‐like responses in aetiolated soybean seedlings at their normal growth temperature of 28 °C. Northern blot analyses revealed that the expression of HS genes hsp17.5, hsp70 and hsc 70 was induced by ethanol. Radioactive amino acids were preferentially incorporated into high molecular weight (HMW) HSPs rather than class I low molecular weight (LMW) HSPs during non‐lethal ethanol treatments. Immunoblot analysis confirmed that no accumulation of class I LMW HSPs occurred after non‐lethal ethanol treatment. Pre‐treatment with a non‐lethal dose of ethanol did not provide thermotolerance, as the aetiolated soybean seedlings could not survive a subsequent heat shock of 45 °C for 2 h. In contrast, non‐lethal HS pre‐treatment, 40 °C for 2 h, conferred tolerance on aetiolated soybean seedlings to otherwise lethal treatments of 7·5% ethanol for 8 h or 10% ethanol for 4 h. These results suggest that plant class I LMW HSPs may play important roles in providing both thermotolerance and ethanol tolerance.     

Transmission of Three Viroids Through Seed and Pollen of Tomato Plants

The severe strain of potato spindle tuber viroid (s-PSTV) as well as chrysanthemum stunt (CSV) and cucumber pale fruit (CPFV) viroids were found to be transmitted through seed and pollen of the tomato cvs. Rutgers and Najwcze?niejszy. Plants pollinated with a pollen infected with any of these three viroids became systematically infected. Plant, fruit and seed symptoms of viroid infection were noted on sap- and pollen-inoculated plants and the yield of these plants was reduced. Tomato cv. Rutgers plants grown from infected seeds were symptomless although all three viroids were detected in these plants by bioassay and by electrophoresis on 5% polyacrylamide gel. When DNA complementary to s-PSTV RNA was used for a direct viroid detection in seed samples by spot hybridization technique it hybridized not only with s-PSTV RNA but also with CSV RNA as well as with CPFV RNA.     

Murine oncornavirus high-molecular-weight RNA structure thermal stepwise dissociation of 70S murine leukemia-sarcoma virus to subunits and low-molecular-weight associated RNAs.

The thermal dissociation into subunits and low-molecular-weight (LMW) associated RNAs of the aggregate structure of 70S RNA of a murine leukemia sarcoma viral complex was studied. By polyacrylamide-agarose gel electrophoresis, it was found that at low temperature a fraction of the genome was converted into an intermediate population of RNA (Im.P) with an apparent molecular weight of 6.6 times 10-6. At higher temperature, the 70S RNA and the Im.P RNA were successively dissociated into two RNA subunits called "I" and "II" and 70S-associated LMW RNAs. The apparent molecular weight of subunit I was about 5 times 10-6 and that of subunit II was about 3.2 times 10-6. The release of 4S, 5S, 5.5S, and 8S RNAs from 70S RNA at various temperatures was studied by composite polyacrylamide gel electrophoresis. It was found that the nature of hydrogen bonding to the 70S RNA was different for each LMW RNA species. A possible relationship of the association between the subunits and each 70S-associated LMW RNA, based on their T-m values, is discussed.     

Direct Isolation of High-Quality Low Molecular Weight RNA of Pear Peel from the Extraction Mixture Containing Nucleic Acid

Low molecular weight RNA (LMW RNA) is generally obtained either from the total RNA or from total nucleic acids solution. Many steps and chemical reagents are involved in traditional methods for LMW RNA isolation where degradation of LMW RNA often occurs, especially for plant materials with high levels of secondary catabolites. In this study, an efficient method was developed to directly isolate pure LMW RNA from pear peel, a material rich in polyphenolics that is covered with a layer of wax. The method was based on polyethylene glycol (PEG) precipitation combining CTAB buffer which is often used to isolate RNA from polysaccharide-rich and polyphenolics-rich materials. The entire procedure could be completed within 6 h and many samples could be processed at the same time. Few and common chemicals are used with this method. Hence, it could be used as an ordinary method in the laboratory. The developed method was further tested by isolating LMW RNA from Arabidopsis. Using the isolated LMW RNA samples, microRNAs were successfully detected and characterized.     

Viroid RNA systemic spread may depend on the interaction of a 71-nucleotide bulged hairpin with the host protein VirP1

Viroids are noncoding circular single-stranded RNAs that are propagated systemically in plants. VirP1 is a protein from tomato, which is an excellent host for potato spindle tuber viroid (PSTVd), and it has been isolated by virtue of its specific in vitro binding to PSTVd RNA. We report on the specific in vivo interaction of VirP1 with full-length viroid RNA as well as with subfragments in the three-hybrid system. The terminal right domain (TR) of PSTVd was identified as a strong interacting partner for VirP1. A weaker partner is provided by a right-hand subfragment of hop stunt viroid (HSVd), a viroid that infects tomato poorly. We present a sequence and structural motif of the VirP1-interacting subfragments. The motif is disturbed in the replicative but nonspreading R+ mutant of the TR. According to our in vivo and in vitro binding assays, the interaction of this mutant with VirP1 is compromised. We propose that the AGG/CCUUC motif bolsters recognition of the TR by VirP1 to achieve access of the viroid to pathways that propagate endogenous RNA systemic signals in plants. Systemic trafficking has been suggested for miRNA precursors, of which the TR, as a stable bulged hairpin 71 nt long, is quite reminiscent.