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1、山东农业大学 硕士学位论文 花生维生素E合成关键酶基因（VTE3、HPT）的克隆、序列分析 与-tmt基因的转化研究 姓名：郭安强 申请学位级别：硕士 专业：作物遗传育种 指导教师：万勇善 2011-06-01 山东农业大学硕士学位论文 1 中 文 摘 要 维生素 E 是一种脂溶性抗氧化剂，对动、植物和人体都有非常重要的生理作用。 然而天然维生素 E 只能由光合细菌和绿色植物合成，人体必须从外界摄取。植物油是 人体摄取维生素 E 的主要途径。花生作为重要的油料作物，是人体所需维生素 E 的重 要来源之一。本研究基于花生 EST 序列，结合 RT-PCR 技术以及以基因组 DNA 为模 板的 P
2、CR 扩增方法，首次从花生中克隆了维生素 E 合成途径的关键酶基因 2-甲基-6- 植基-1,4-苯醌甲基转移酶基因（VTE3）和尿黑酸植基转移酶基因（HPT） ，并且分别克 隆了 13 个不同类型花生栽培品种和 6 个二倍体野生种的 VTE3 DNA 序列，在比较各 序列同源性的基础上，对不同类型花生栽培品种是否有相同起源问题以及花生栽培种 与各野生种之间的亲缘关系进行了分析探讨；利用农杆菌介导法将拟南芥 -维生素 E 甲基转移酶基因（-tmt）转入花生栽培品种得到 T0代和 T1代阳性转化植株。取得的 主要研究结果如下： （1）从丰花 2 号、兰娜 1 号和荔浦大花生 3 个栽培品种中分别
3、克隆到 2 条 VTE3 cDNA 片段，命名为 rVTE3-1 和 rVTE3-2。品种间相应序列同源性为 100%。rVTE3-1 和 rVTE3-2 的编码区长 1059 bp， 均编码 351 个氨基酸， 二者核苷酸序列同源性 97.8%， 存在 10 个变异位点，其中 8 个为 SNP 变异；氨基酸序列同源性 98.6%，存在 5 个氨 基酸差异。 （2）从花生栽培品种 Krapt.st.16 中克隆得到一条 HPT cDNA 片段，长 1269 bp， 命名为 AhHPT。AhHPT 最大开放阅读框为 1230 bp，编码含有 409 个氨基酸残基，与 大豆、 苜蓿、 木薯、 拟南
4、芥等16种植物的HPT蛋白序列同源性较高， 为61.46% 79.71%。 （3）从 13 个栽培品种分别克隆得到 2 条 VTE3 DNA 片段，命名为 gVTE3-1 和 gVTE3-2。gVTE3-1 在 13 个品种间核苷酸序列同源性为 99.9%, gVTE3-2 核苷酸序列同 源性为 100%。丰花 2 号 gVTE3-1 和 gVTE3-2 同源性 96.6%，内含子存在 36 个 SNP 位 点和 3 个限制性内切酶识别的多态性位点。从 6 个二倍体野生花生分别克隆得到一条 VTE3 DNA 片段， A 染色体组 4 个野生种以及 B 染色体组野生种 A. batizocoi
5、的 gVTE3 同源性达 99.9%以上，仅有 2 个碱基的差异。B 染色体组野生种 A. ipaensis 的 gVTE3 与其余野生种 gVTE3 同源性相对较低，为 97.0%。A. ipaensis 的 gVTE3 与丰花 2 号 gVTE3-2 同源性 100%；丰花 2 号 gVTE3-1 与 A 染色体组野生种的 gVTE3 同源性相对 较高，最高为 98.7%。进化树分析显示，丰花 2 号的 gVTE3-2 与 A. ipaensis 的 gVTE3 花生维生素 E 合成关键酶基因（VTE3、HPT）的克隆、序列分析与 -tmt 基因转化研究 2 聚为一组；A 染色体组 4 个
6、野生种和 B 染色体组野生种 A. batizocoi 的 gVTE3 以及丰 花 2 号 gVTE3-1 聚为一组。 （4）得到 164 株具有 PPT 抗性并且 PCR 检测目的基因（-tmt）呈阳性的转基因 植株，经扩繁后移栽成活 T0代单株 560 个，获得其 T1代种子；获得来自 34 个株系 T1 代单株 356 个，经 PCR 检测其中 185 个植株呈阳性，获得其 T2代种子。 关键词：花生；维生素 E；基因克隆；遗传转化 山东农业大学硕士学位论文 3 Cloning and sequence analysis of vitamin E synthesis-related ke
7、y enzyme genes (VTE3 and HPT) in Arachis species and genetic transformation of -tmt Abstract Vitamin E is an important class of lipid-soluble compounds with antioxidant activities that plays a very important role in plant, animal and human being. However, natural vitamin E is synthesized only by oxy
8、genic photosynthetic organisms, which can not synthesized by human body. Plant oil is the main resource of the vitamin E in human nutritious. Peanut (Arachis hypogaea L.) is an important oilseed crop, which is also one of the important source of vitamin E human body needed. Based on EST sequences, w
9、e cloned vitamin E synthesis-related key enzyme gene of 2-methyl-6-phytyl-1, 4-benzoquinone methyltransferase (VTE3) and homogentisate phytyltransferase (HPT) in peanut by RT-PCR for the first time. Two full-length DNA of VTE3 were cloned from each of thirteen varieties of A. hypogaea and one was cl
10、oned from each of six wild species by PCR. Based on these VTE3 DNA sequences, we studied the origin of different type varieties of peanut and the genetic relationship between cultivated peanut and each wild species involved. Whats more, the -tocopherol methyltransferase gene (-tmt) was introduced in
11、to peanut cultivar and T0 and T1 positive transgenic seedlings were obtained. The major results are abstracted as follows: (1) Two cDNA sequences of VTE3 were cloned from each cultivar of Fenghua 2, Lanna 1 and Lipudahuasheng, which are identical and were symbolized rVTE3-1 and rVTE3-2. Both of the
12、sequences code 351 amino acids, which share 97.8% identity in nucleotide acid level and share 98.6% identity in amino acid level. Eight sites of SNP variations were identified between rVTE3-1 and rVTE3-2. Five amino acids differences were identified between the two relevant amino acid sequences. (2)
13、 One HPT cDNA sequence, 1269 bp in length and symbolized AhHPT, was cloned from cultivar Krapt.st.16. The largest open reading frame of AhHPT gene has 1230 bp in length and encoded a protein of 409 amino acid residues. Blast result showed that the deduced amino acid sequence of AhHPT is highly homol
14、ogous with the HPT amino acid sequences from sixteen plants include Glycine max, Medicago sativa, Manihot esculenta, Arabidopsis thaliana, et al. (3) Two DNA sequences of VTE3, symbolized gVTE3-1 and gVTE3-2, were cloned from 花生维生素 E 合成关键酶基因（VTE3、HPT）的克隆、序列分析与 -tmt 基因转化研究 4 each of thirteen cultivar
15、s. The thirteen sequences of gVTE3-1 from the thirteen cultivars share 99.9% identity in nucleotide acid level and the thirteen sequences of gVTE3-2 are identical. A total of 36 sites of SNP variations and three sites of endonuclease recognition variations were identified between the intrones of seq
16、uences gVTE3-1 and gVTE3-2 from Fenghua 2, which share 96.6% identity in nucleotide acid level. One DNA sequences of VTE3 was cloned from each of six wild diploid species. The gVTE3 sequences of four wild diploid species from A genome and the sequences of gVTE3 from A. batizocoi share more than 99.9% identity in nucleotide acid level with only two bases differences. The sequence of gVTE3 from A. ipaensis and the sequence of gVTE3-2 from Fenghua 2 are identical. The sequence of gVTE3
