桃潜隐花叶类病毒花叶分离物的分子和生物学特性分析

Molecular and biological characteristics of peach latent mosaic viroid mosaic isolate

桃潜隐花叶类病毒(peach latent mosaic viroid,PLMVd)是桃树上的重要类病毒。PLMVd侵染桃树后会引起花叶、黄化和白化等不同的叶片症状。目前,PLMVd侵染引发花叶的机制不明。本文克隆了来自我国田间油桃样品的花叶(M)分离物和无症状(N)分离物的86条PLMVd全长序列,大小为336~338 nt。用DnaSP 5.0对克隆的序列进行分析,获得31条单体型(变体)序列,来自M分离物的单体型多样性(Haplotype diversity)较低为0.79,N分离物较高为0.90。与无症状的N分离物序列相比,M分离物序列变异主要发生在全序列的五个核苷酸区,来自M分离物的MY1单体型序列为优势序列,与少数其他序列变体聚在系统发育树的Group I,并与已报道的典型白化P1.148分离物序列相似性为89.4%。通过PLMVd侵染性克隆和基因合成的构建、接种方法的比较,构建了有侵染性的PLMVd二倍体和有效接种技术。构建的PLMVd MY1二倍体cDNA质粒,通过强化茎切接种法接种,能系统侵染山毛桃并表现典型的花叶症状。研究结果为进一步研究PLMVd引发花叶的分子机制奠定了基础。

Peach latent mosaic viroid(PLMVd)is an important viroid infecting peach trees.PLMVd can cause various leaf symptoms including mosaic,yellowish and calico,etc.At present,the mechanism of PLMVd infection causing mosaic leaves is unknown.In this study,86 full-length PLMVd sequences,in size of 336-338 nt,were isolated and cloned from the mosaic(M)and asymptomatic(N)leaves of nectarine trees collected in the field in China.By DnaSP 5.0 analysis,31 haplotype(variant)sequences were obtained in the cloned sequences.The haplotype diversity(Hd)was relatively low as 0.79 for M isolates,while was high as 0.90 for N isolates.Compared with N isolates,sequence analysis showed that the sequence variation of M isolates mainly occurred in five regions of the whole sequence.MY1 haplotype sequence was dominant among those of M isolates,which clustered in phylogenetic group I with a few other variants and shared 89.4%identity with that of P1.148,a typical peach calico isolate.Through a comparative study of PLMVd cDNA infectious clones,gene synthesis and inoculation methods,the infectious PLMVd diploid cDNA clone construction and effective inoculation methods were established.The constructed MY1 diploid cDNA recombinant plasmid can infect the Prunus davidiana Franch systematically and exhibit typical mosaic symptoms after high-stressed stems slashing inoculation.The results will build a foundation for further study about the molecular mechanism of PLMVd inducing peach mosaic symptom.