Pk-αts高表达提高红松对松材线虫耐病性

High Expression of the Pk-αts Increased the Tolerance of Pinus koraiensis against Bursaphelenchus xylophilus

松材线虫的北扩已经在东北局域引发毁灭性灾害,对东北林区顶级生态群落的重要组成树种红松构成重大威胁。对丰富红松与松材线虫互作分子机理的研究,为耐病红松选育奠定理论基础。人工接种鉴定20个家系红松对松材线虫的耐病性差异,并将不同家系红松分为耐病、感病和高感3类。提取耐病和高感红松核糖核酸(RNA)进行转录组测序,筛选并克隆红松耐病相关的α-松油醇合酶基因Pk-αts。RT-qPCR随机检测4个耐病、2个感病和4个高感红松Pk-αts表达量,计算此10个家系红松相对耐病指数,分析Pk-αts表达量与红松相对耐病指数的相关性。结果表明,接种松材线虫20 d后耐病红松仅数根针叶褪绿,平均相对耐病指数为0.88,而高感红松表现出局部松针变红,平均相对耐病指数为0.09。转录组鉴定到在耐病红松差异上调表达且在高感红松无变化或下调表达的基因1770个,其中Pk-αts在耐病红松的相对表达量为2.83,极显著,但在高感红松中相对表达量为-1.27。基因克隆得到Pk-αts长度为1230 bp。该基因编码蛋白质由409个氨基酸构成,等电点为5.55,相对分子质量为47.60 ku,经比对后与α-松油醇合酶基因同源性最高,且含有植物单萜烯合成酶DDxxD、RRx 8 W保守基序及Terpene_synth等结构域,符合单萜烯合成酶结构特征。RT-qPCR检测耐病红松的Pk-αts基因表达上调(平均表达量为1.39),感病和高感红松Pk-αts基因不变或下调表达,表明在侵染早期,Pk-αts基因的上调表达与红松耐病时空同步。Pk-αts表达量与红松相对耐病指数显著正相关(皮尔逊系数为0.911,P<0.01),表明Pk-αts基因的上调表达提高了红松耐病性。综上所述,不同家系红松对松材线虫耐病性不同,部分红松具有一定耐病性,Pk-αts基因上调表达减轻了发病症状,有助于提高红松对松材线虫的耐病性。以Pk-αts基因作为检测靶标可用于红松耐病性鉴定,有助于耐病红松选育。

The northward expansion of Bursaphelenchus xylophilus has caused devastating disasters in the northeastern region,posing a significant threat to Pinus koraiensis,which is the major component species of the top ecological community of the Northeast China forest area.This study aims to enrich the molecular mechanism of the interaction between P.koraiensis and B.xylophilus,thus laying a theoretical foundation for the breeding of disease-tolerant P.koraiensis.The disease tolerance of 20 families of P.koraiensis to B.xylophilus was identified by artificial inoculation and different families of P.koraiensis were divided into three categories:tolerant,susceptible and high-susceptible.The RNA of tolerant and high-susceptible P.koraiensis was extracted for transcriptome sequencing.Theα-terpineol synthase gene(Pk-αts)related to disease tolerance was screened and cloned.The expression level of Pk-αts in 4 tolerant,2 susceptible,and 4 high-susceptible P.koraiensis was randomly detected by RT-qPCR,and the relative tolerance index of these 10 P.koraiensis families was calculated.The correlation between Pk-αts expression level and the relative tolerance index of P.koraiensis was analyzed.The results showed that after inoculating B.xylophilus for 20 days,only a few needles of tolerant P.koraiensis turned yellow,with an average relative tolerance index of 0.88,while high-susceptible P.koraiensis showed partial needle redness,with an average relative tolerance index of 0.09.Transcriptome sequencing identified 1770 genes that were up-regulated in tolerant P.koraiensis and had no change or down-regulated in high-susceptible P.koraiensis.The relative expression of Pk-αts in tolerant P.koraiensis was 2.83,which was extremely significant,but the relative expression in high-susceptible P.koraiensis was-1.27.The cloned Pk-αts gene was 1230 bp in length,encoding a protein composed of 409 amino acids,with an isoelectric point of 5.55 and a relative molecular weight of 47.60 ku.After comparison,it had the highest homology with theα-terpineol synthase gene and contained conserved motifs such as DDxxD and RRx8W of plant monoterpene synthases and Terpene_synth domain,which were consistent with the structural characteristics of monoterpene synthases.RT-qPCR detection showed that the expression of the Pk-αts gene was upregulated(the average expression level is 1.39)in tolerant P.koraiensis,while it was unchanged or downregulated in susceptible and high-susceptible P.koraiensis,indicating that the upregulation of the Pk-αts gene expression was synchronized with the temporal and spatial of P.koraiensis tolerance to in the early stage of B.xylophilus infection.The expression level of Pk-αts was positively correlated with the relative tolerance index of P.koraiensis(Pearson's coefficient was 0.911,P<0.01),indicating that the up-regulated expression of the Pk-αts gene improved the disease tolerance of P.koraiensis.In summary,different families of P.koraiensis have different tolerance to B.xylophilus,and some P.koraiensis have certain disease tolerance.The up-regulated expression of Pk-αts gene alleviates disease symptoms and helps to improve the disease tolerance of P.koraiensis to B.xylophilus.The Pk-αts gene can be used as a detection target for identifying tolerance of P.koraiensis to B.xylophilus,which is helpful for the breeding of tolerant P.koraiensis.