除虫菊TcALDH和TcGLIP基因启动子克隆及功能分析

Cloning and functional analysis of promoter of TcALDH and TcGLIP genes in Tanacetum cinerariifolium

关键词:  除虫菊, 醛脱氢酶(TcALDH), GDSL脂肪酶(TcGLIP), 启动子, 功能分析

Key words:  Tanacetum cinerariifolium, TcALDH, TcGLIP, promoter, functional analysis

摘要:

天然除虫菊酯是从除虫菊(Tanacetum cinerariifolium)中提取的绿色植物源生物杀虫剂。醛脱氢酶(TcALDH)和GDSL脂肪酶(TcGLIP)是除虫菊酯生物合成途径中的关键限速酶。为探究TcALDH和TcGLIP基因的功能,该研究从除虫菊无性系‘W99'中克隆得到TcALDH和TcGLIP基因的启动子,并通过生物信息学分析、组织化学染色(GUS染色)、荧光素酶报告实验和外源植物激素处理实验对其启动子的调控元件、启动子活性、激素诱导特异性和组织特异性进行分析。结果表明:(1)克隆得到的TcALDH和TcGLIP启动子序列分别为2 848、1 343 bp,均含有多个与逆境应答和激素信号相关的顺式作用元件。(2)分别构建了启动子和荧光素酶融合的植物表达载体,在烟草叶片中观察荧光成像发现,TcALDH启动子具有茉莉酸甲酯(MeJA)和脱落酸(ABA)激素诱导特异性。(3)用MeJA和ABA处理除虫菊‘W99'组培苗发现,TcALDH的表达量在12 h内受ABA诱导时上调,受MeJA诱导时先升高后降低,TcGLIP的表达量受ABA和MeJA诱导下调。(4)分别构建了TcALDH和TcGLIP启动子与GUS基因融合的植物表达载体,转化烟草并对其转基因叶片进行GUS活性染色发现,TcALDH启动子在烟草叶片腺体、腺毛头部及叶肉细胞中表达,而TcGLIP启动子仅在烟草叶肉细胞中表达。综上认为,TcALDH和TcGLIP的启动子具有组织特异性,TcALDH启动子具有MeJA和ABA激素诱导特性。该研究结果为除虫菊TcALDH和TcGLIP基因参与除虫菊酯合成的调控机制提供了新见解。

Abstract:

Natural pyrethrin is a green botanical insecticide that extracted from the aboveground tissues of pyrethrum(Tanacetum cinerariifolium). Aldehyde dehydrogenase(TcALDH)and GDSL lipase(TcGLIP)are key rate-limiting enzymes involved in pyrethrin biosynthesis pathway in pyrethrum. The promoters of TcALDH and TcGLIP genes were cloned from the genomic DNA of pyrethrum clone ‘W99' in order to investigate the regulatory mechanism of these genes. The regulatory elements, activity, hormone specificity and tissue inducibility of the two promoters were analyzed through bioinformatics analysis, histochemical staining(GUS staining), luciferase reporting, and exogenous hormone treatment. The results were as follows:(1)Using pyrethrum genomic DNA as a template, specific primers were used to clone the pTcALDH and pTcGLIP fragments. The sequence lengths of pTcALDH and pTcGLIP were 2 848 and 1 343 bp, respectively, and the promoter analysis software the PlantCARE predicted that they both contained multiple cis-elements related to stress response and hormone signals.(2)The plant expression vectors fused by pTcALDH and pTcGLIP and luciferase report gene were constructed, and were transformed into tobacco(Nicotiana benthamiana)to analyse hormone inducibility by observing the fluorescence imaging in tobacco leaves. The results demonstrated that the pTcALDH displayed typical hormone inducibility of methyl jasmonate(MeJA)and abscisic acid(ABA), whereas the pTcGLIP showed no response.(3)The tissue culture seedlings of pyrethrum ‘W99' were treated with MeJA and ABA, the expression of TcALDH was up-regulated by ABA within 12 h, and first increased and then decreased under MeJA treatment; the expression of TcGLIP was down-regulated by ABA and MeJA.(4)We constructed the expression vectors of pTcALDH and pTcGLIP fused with GUS reporters and transformed them into tobacco, then the transient transformation of tobacco drived the expression of GUS gene and showed initiating activity. It was found that the pTcALDH expressed in both the glands, glandular hair heads and mesophyll of the leaves, while the pTcGLIP was only expressed in the parenchyma cell. These results indicated that the pTcALDH and pTcGLIP were tissue-specific promoters, and the pTcALDH appeared MeJA-inducible and ABA-inducible characteristics. This study provides a new insight into the regulatory mechanism of TcALDH and TcGLIP genes involved in pyrethrin synthesis.