应用抑制性差减杂交技术研究枣缩果病病程相关基因  

Identification of differentially expressed genes related with Jujube fruit shrink disease by suppression subtractive hybridization

作　　者：焦小利 杨卉芯[1] 宋晓斌[1] 

机构地区：[1]西北农林科技大学林学院,陕西杨凌712100

出　　处：《果树学报》2017年第12期1520-1526,共7页Journal of Fruit Science

基　　金：基金项目:红枣裂果防控及高效栽培技术研究与示范(HZKY-01)

摘　　要：【目的】了解细交链孢菌侵染枣果实过程的差异表达基因。【方法】以白熟期‘蜂蜜罐’枣的果实为材料,人工接种细交链孢菌,分别在接种0.5、1、2、3和4 d后取样,通过抑制性消减杂交技术(suppression subtractive hybridization,SSH)构建了病原菌诱导的差异表达的c DNA消减文库。【结果】PCR鉴定随机挑取的阳性克隆,显示插入片段大小为200~900 bp。随机挑选200个阳性克隆进行测序,利用BLASTx在Gen Bank Nr数据库进行序列比对分析,共获得118个Unigenes。对这些ESTs进行功能分类发现细交链孢菌侵染下诱导表达基因涉及植物细胞内的多种代谢和应答过程,其中包括抗病/防御类、信号传导途径类、新陈代谢类、蛋白质合成和加工类及细胞结构的组成等,尤其以抗病/防御类基因所占比例最大(27.17%)。【结论】通过SSH研究了缩果病病原侵染果实过程的差异表达基因,鉴定到一些与枣缩果病相关的基因,为进一步研究相关基因及今后筛选防治枣缩果病提供理论基础。[Objective] Jujube fruit shrink disease caused by Alternaria alternate is one of main fruit dis- eases of Ziziphus jujuba. It could reduce the yields and quality of jujube severely. Although the disease has been investigated at the tissue and physiological level, the molecular response of jujube fruits to A. alternate infection is still unclear. Investigation on the differentially expressed genes can help us better understand the molecular processes involved in the interactions between pathogen and jujube fruits. In this study, a suppression subtractive hybridization （SSH） technique was used to identify the differentially ex- pressed genes in jujube fruits infected by A. alternate in order to illustrated the molecular response of ju- jube fruits to the disease. [Methods] The physiological race （ZS091） of A. alternata was provided by Henan Academy of forestry, China. The isolate was cultured on PDA medium for one week and the prolif- erated spores were dissolved in Tween-80, and adjusted to 108 conidia per mL in distilled water as the inoculum. The inoculum was artificially inoculated on the fruits of Z. jujuba ＇ Fengmiguan＇ in the white ma- ture stage in the Jujube Experimental Station of Northwest A ＆ F University, Qingjian county, Shaanxi province, China. The fruits inoculated with distilled water were set as the reference. We picked three fruits 0.5, 1, 2, 3 and 4 d after inoculation, respectively. The fruit samples were immediately frozen in liquid nitrogen, and transported to laboratory in dry ice and stored at- 80℃ before RNA extraction. Total RNA was extracted with the ＇MiniBEST Plant RNA Extraction Kit＇ reagent （Takara） according to manufacturer＇ s instruction, cDNA subtractive library on differential expression induced by the infection of A. alternate by SSH was constructed with PCR-SelectTM cDNA Subtraction Kit （Clontech）. Double-stranded cDNAs of tester （the pooled samples of A. ahernata-inoculated fruits） and driver （non-inoculated fruits） were synthesized from 1

关 键 词：枣缩果病 抑制差减杂交技术 表达序列标签 

分 类 号：S665.1[农业科学—果树学]