摘要
【目的】建立并优化乙烯利调控甘蔗基因差异表达的cDNA-AFLP分析体系和银染体系,利用该技术对乙烯利处理的甘蔗进行基因差异表达研究,探讨乙烯利调控甘蔗生长的分子机理。【方法】以甘蔗品种ROC16为材料,在甘蔗生长40d左右时,叶面喷施200mg/L乙烯利溶液(对照为清水)0、3、6、12、24、48h后,分别取甘蔗叶片提取总RNA,建立cDNA-AFLP分析体系和银染体系;利用193对引物组合对乙烯利处理和对照的甘蔗叶片RNA混合池的样品进行差异表达分析,对部分差异转录片段进行回收、克隆、反向Northern杂交验证、序列分析和功能分析等。【结果】经cDNA-AFLP体系反应扩增,乙烯利处理和对照每个样品的扩增条带数均在35～80之间,处理和对照之间条带多态性明显。在反向Northern杂交验证分析中,24个测试样品中有11个无差异,假阳性率高达46%;在乙烯利作用下,甘蔗的CHI、GST、ARP、LHC、核结合蛋白基因以及一批未知基因差异表达,其中CHI、GST、LHC和核结合蛋白基因是与促进植物的抗病抗逆、提高光合作用等密切相关的因子。【结论】cDNA-AFLP技术是研究乙烯利诱导甘蔗相关基因差异表达和调控的一种较有效方法,具有可行性。乙烯利可能是通过调控甘蔗体内GST、CHI、ARP和LHC等基因的表达,从而表现出促进甘蔗生长、提高光合作用、增强抗病抗逆性等生理效应。
[Objective ]The present experiment was conducted to develop the optimized cDNA-AFLP protocol and an efficient silver staining system to study the differential expressions of genes regulated by ethephon, and to understand the molecular mechanism of ethephon regulated growth and development in sugarcane. [Method]Forty-day old plants of sugarcane variety ROC16 were foliarly sprayed with ethephon （200 nag/L）, and the leaves were sampled at 0,3,6,12,24 and 48 hours to extract the total RNA and establish the cDNA-AFLP protocol and silver staining system. Using combinations of 193 primer pairs, some transcript derived fragments （TDFs） were isolated, cloned, detected with reverse Northern blotting and sequenced. [Result]The cDNA-AFLP analysis results showed high level of polymorphism amongst the treatment and control. 35 to 80 bands were amplified in each sample. Eleven samples with 24 TDFs did not display any differential expression, and the ratio of hypocrite positive TDFs was recorded as 46%. The ehitinase I（CHI）,glutathione S-trans- ferase （GST）, auxin-responsive protein （ARP）, light harvesting chlorophyll a/b-binding protein （ LHC）, nuclear binding protein gene and a set of unknown genes were differentially expressed in the ethephon treatments. CHI and GST have an important role in resistance to stress and various diseases,while LHC and nuclear binding protein are related to light harvesting and CO2 fixation in photosynthesis, respectively. [Conclusion ]The cDNA-AFLP technique was found to be very ef- fective and feasible to study the differential expression of genes in sugarcane. Ethophen was found to regulate the expression of genes related to the primary metabolism of sugarcane and resistance to disease and stress by regulating the expression of related genes.
出处
《南方农业学报》
CAS
CSCD
2011年第1期6-10,共5页
Journal of Southern Agriculture
基金
国家科技支撑计划项目(2007BAD30B02)
广西科技基础条件平台建设项目(09-007-08)
广西科学研究与技术开发计划项目(桂科能0815011-6-1)
广西科学基金项目(桂科基0778006-6)
广西科学基金项目应用基础研究专项项目(桂科基0991021)
广西农业科学院科技发展基金项目(2007022)
作者简介
魏源文（1974-），男，广西岑溪人，博士，副研究员，主要从事基因克隆与转基因研究工作。
通信作者，E-mail：liyr@gxaas．net.
