摘 要

鹅掌楸属(Liriodendron)为木兰科树种，现仅存2种，即北美鹅掌楸（Liriodendron tulipifera）和鹅掌楸(Liriodendron chinense)。北美鹅掌楸适应性强，广泛分布于北美大陆东部，而鹅掌楸仅星散分布于我国长江流域及越南北部。植物GSTs家族基因可响应多种非生物胁迫，包括脱水、UV、冷害、干旱、高盐以及ABA等，可调节植物的抗逆性，因而目前学界普遍认为GST基因家族的功能与植物适应性有关。

以鹅掌楸为试验材料，从北美鹅掌楸和鹅掌楸转录组数据库中挖掘出4条GST基因家族的EST序列。以这4条GST基因家族的EST序列为基准设计引物，通过反转录PCR (RT-PCR)与cDNA末端快速扩增 (RACE) 技术从北美鹅掌楸中克隆得到4条长为1150 bp、932bp、1022bp、1067bp的基因全长序列，分别命名为LtGSTU1、LtGSTZ1、LtGSTF1、LcGSTT1。这四种蛋白质分子量分别约为：25kDa、25kDa、24kDa、28kDa；蛋白质数目分别为：3566aa、3608aa、3457aa、3996aa；理论等电点pI分别为：5.60、6.53、6.66、9.20；根据ProtParam的分析结果判断这4个蛋白均为亲水性蛋白。根据Guruprasad方法表明LtGSTU1、LtGSTZ1的蛋白稳定，LtGSTF1、LcGSTT1的蛋白不稳定。α-螺旋(Alpha helix)是这四个蛋白质的主要二级结构。采用同源模建的方法对该基因编码蛋白的三级结构进行了预测。

实时荧光定量PCR分析结果表明，该4个基因在各个组织中均有表达。其中，LtGSTU1和LtGSTF1在叶中的表达量最高。LtGSTZ1在花器官中的表达量最高，在叶中的表达量最低。LcGSTT1在叶中表达量最高，在茎和叶芽中的表达量偏低。研究结果可为进一步分析鹅掌楸GSTs的功能奠定基础。

关键词：鹅掌楸；GSTs；克隆；序列分析；表达

The Cloning and Expression Analysis of GSTs Gene in Liriodendron chinense

ABSTRACT

Liriodendron is a genus of Magnoliaceae, now only 2 species, L. tulipifera and L. chinense. L. tulipifera is highly adaptable and widely distributed in the eastern part of the North American continent, while L. chinense is scattered only in China's Yangtze River Basin and northern Vietnam. Plant GSTs family genes can respond to a variety of abiotic stress, including dehydration, UV, chilling injury, drought, high salt ABA and its like. Because of their function of adjusting plant resistance, the GST family is well known for their relating to plant adaptability.

The EST sequences of four GST gene families were extracted from the database of L. tulipifera and L. chinense. The primers were designed based on the EST sequences of the four GST gene families. A total of 1150bp, 932bp, 1022bp and 1067bp genes were cloned from L. tulipifera by reverse transcription PCR and cDNA rapid amplification, named LtGSTU1, LtGSTZ1, LtGSTF1, LcGSTT1, separately. The molecular weights of these four proteins were about 25kDa, 25kDa, 24kDa and 28kDa, respectively; the number of proteins was 3566aa, 3608aa, 3457aa and 3996aa respectively, and the theoretical isoelectric point pI were 5.60,6.53,6.66,9.20; the four proteins are hydrophilic protein according to the analysis of ProtParam results. According to the Guruprasad method, Lt GSTU1, Lt GSTZ1 protein was stable and Lt GSTF1, LcGSTT1 protein was unstable. The alpha helix is the primary secondary structure of these four proteins. The third structure of the protein was predicted by homology modeling.

The four genes were expressed in all tissues by real-time fluorescence quantitative PCR. This may be related to its resistance to stress. The expression of LtGSTU1 and LtGSTF1 in leaves was the highest. LtGSTZ1 had the highest expression level in floral organs and the lowest expression in leaves. LcGSTT1 had the highest expression level in leaves and lowest expression in stem and leaf buds. The results could lay a foundation for the further analysis of the function of GSTs.

Keywords: Liriodendron , GSTs, cloning, sequence analysis, expression目录

1 引言 - 5 -

1.1鹅掌楸简介 - 5 -

1.2谷胱甘肽转移酶研究现状 - 5 -

1.2.1谷胱甘肽转移酶功能 - 6 -

1.2.2 GST基因的表达 - 6 -

1.3 研究目的及意义 - 7 -

2 材料和方法 - 8 -

2.1实验材料 - 8 -

2.1.1植物材料 - 8 -

2.1.2菌株和载体 - 8 -

2.1.3培养基的配制 - 8 -

2.1.4实验试剂 - 8 -

2.2仪器设备 - 9 -

2.3实验方法 - 9 -

2.3.1鹅掌楸RNA的提取与质量鉴定 - 9 -

2.3.1.1试剂盒法提取植物RNA - 9 -

2.3.1.2总RNA的质量鉴定 - 10 -

2.3.2鹅掌楸GST基因中间片段的扩增 - 10 -

2.3.3鹅掌楸GST基因RACE扩增 - 12 -

2.3.4目的基因的克隆和转化 - 14 -

2.3.5生物信息学分析 - 15 -

2.3.6 GSTs基因组织表达分析 - 15 -

3 结果与分析 - 16 -

3.1 GST基因的克隆 - 16 -

3.2 GST基因的序列分析 - 18 -

3.2.1鹅掌楸GST基因的同源序列比对 - 18 -

3.3蛋白质序列的基本性质分析 - 20 -

3.3.1理化性质分析 - 20 -

3.3.2疏水性分析 - 21 -

3.3.3跨膜区分析 - 23 -

3.3.4信号肽预测 - 25 -

3.3.5亚细胞定位 - 27 -

3.4结构域分析 - 28 -

3.5空间结构预测和分析 - 28 -

3.5.1蛋白质二级结构预测 - 28 -

3.5.2蛋白质三级结构预测 - 29 -

3.6 Lt GST基因的组织特异性表达分析 - 30 -

3.7讨论 - 31 -

致 谢 - 32 -

参考文献 - 33 -

1 引言

1.1鹅掌楸简介

鹅掌楸属(Liriodendron)是双子叶植物纲中比较原始的木兰亚纲木兰科鹅掌揪亚科的速生高大落叶乔木，现仅存2个种：一种是产于北美的北美鹅掌楸（Liriodendron tulipifera）,另一种是产于我国长江流域及越南北部的鹅掌楸(Liriodendron chinense)^[1]。

北美鹅掌楸是分布在美国中东部，被称为活化石。最近这些年，我国开始引种大量北美鹅掌楸，但由于其价格比较昂贵，并具有深度休眠性，给大量育苗带来不便^[31]。鹅掌楸星散分布于长江以南海拔500~1200ｍ 阔叶林中，比较明显的分界线是浙江青田和云南金平。分部属于我国地形中的第二阶梯。分布地区可划分成西亚及东亚地区，北部过黔北中山、湘西、鄂西，之后可延伸到西南的云贵高原，南部呈云贵高原南坡份至向西南分布到南回归线之南到我国金平、麻栗坡及越南的北部，呈现一条东北至西南过渡带状的分布带，在西部呈现连片分布的状态；在东部呈岛状分布^[2-3]。