KMS XINJIANG INSTITUTE OF ECOLOGY AND GEOGRAPHY，CAS
| 松叶猪毛菜 NADP-ME 家族基因的克隆及其叶绿体型基因的功能研究 | |
| Alternative Title | Cloning of NADP-ME family gene and studying the function of its chloroplast gene |
| 王玉兰 | |
| Subtype | 硕士 |
| Thesis Advisor | 闻志彬 |
| 2020-06-30 | |
| Degree Grantor | 中国科学院大学 |
| Place of Conferral | 北京 |
| Degree Discipline | 理学硕士 |
| Keyword | C3-C4中间型植物 NADP-ME 家族 功能验证 C4光合基因进化 C3-C4 intermediate plant NADP-ME family functional verification evolution of C4 photosynthetic gene |
| Abstract | NADP-苹果酸酶（NADP-ME） 是苹果酸代谢通路中的关键酶， 它不仅参与植物的光合作用， 还参与植物的环境逆境防御及相关代谢活动。 课题组前期的研究表明， C3-C4中间型植物松叶猪毛菜通过增强 C4途径来抵御干旱胁迫， 并且在所研究的光合酶中， 仅有 NADP-ME 活性在不同程度的干旱胁迫下都显著升高，暗示该基因在松叶猪毛菜响应逆境胁迫中具有重要作用。 并且， 植物光合基因NADP-ME 进化关系复杂， 为我们认识 C4光合基因的进化提供了很好的素材。本研究拟克隆松叶猪毛菜 NADP-ME 家族基因， 分析 NADP-ME 组织表达特性及响应逆境胁迫的模式， 并验证松叶猪毛菜叶绿体型 NADP-ME 在非生物胁迫下（光照、 干旱和盐） 的功能； 同时探究 NADP-ME 家族基因在被子植物中的动态演化规律。 主要结果如下：1. 基于松叶猪毛菜的根、 茎和叶混库建立的转录组数据， 本文克隆获得了 3条 NADP-MEs， 命名为 Salchl， Salcyt1 和 Salcyt2。 ①它们的 CDS（编码氨基酸的序列） 长度分别为 1941 bp， 1755 bp， 1758 bp， 编码 646， 584， 585 个氨基酸，这 3 个基因均含有 NADP-ME 的 5 个保守结构域。 ②通过亚细胞定位研究表明，Salchl 编码的蛋白定位于叶绿体， Salcyt1 和 Salcyt2 编码的蛋白均定位于细胞质。③在转录水平上， 这 3 个基因在松叶猪毛菜根、 茎和叶中都有表达， 但没有明显的组织特异性表达模式； 在强光、 干旱和盐胁迫下松叶猪毛菜幼苗根和叶中这 3个 NADP-ME 具有不同基因转录表达方式， 其中 Salchl 的转录表达被这 3 种处理显著诱导， 且在根和叶中有一致性。2. 将 Salchl 稳定转化至拟南芥中进行功能验证。 结果表明： ①正常条件下，过表达 Salchl 能提高转基因拟南芥植株的光合能力。 与 WT 相比， 转基因植株的光系统相关基因（Psb D、 Psb Q、 Psa F、 Psa H） 和捕光色素相关基因（CHL M、Por B、 LHC A2、 LHC H4、 LHC B3） 的表达量、 ETR（光合电子传递速率） 、 Y（II） （实际光能转化效率） 以及叶绿素含量都有明显的增加； ②对 WT 和过表达 Salchl 转拟南芥植株进行强光、 干旱和盐胁迫下的对比研究， 与 WT 相比， 种子萌发阶段， 过表达 Salchl 不能提高植株抗盐、 强光和干旱能力； 幼苗阶段过表达 Salchl 能不同程度的提高拟南芥植株的根长， 鲜重和侧根数目； 在成苗阶段，盐， 干旱和强光胁迫下， 过表达 Salchl 拟南芥的 H2O2含量减少， 脯氨酸含量增加， 表明过表达 Salchl 能提高植株抗逆能力。 盐和强光胁迫下， 转基因植株 ROS酶促系统的 SOD， POD 和 CAT 含量增加； 干旱胁迫下， 转基因植物 ROS 非酶促系统的 ASA/DHA， GSH/GSSG 比值增加。 并且， 在 3 种非生物胁迫下， 转基因植株的光能转化速率及电子传递能力都增加3.基于涵盖被子植物关键节点的 64 个物种的 282 条 NADP-ME 序列， 并以藻类、 苔藓、 蕨类和裸子植物的 10 个物种的 28 条 NADP-ME 序列为外类群， 从系统发育和序列特征入手， 利用生物信息学手段探究 NADP-ME 家族在被子植物中的演化历史。 结果表明： ①单/双子叶植物的 NADP-MEs 在基因复制/丢失事件、基序组成和分布上均存在差异。 ②与 NADP-ME 的 5 个保守域相比， 胞质型NADP-ME 序列在每个保守区域都发生许多变化。 但质体型 NADP-ME 只在保守区 II 的保守序列中存在较多的变化。 ③4 条藻类植物序列位于外类群基部且与其他外类群序列区分开来。 将这些被子植物的 NADP 分为 5 个类型， 包括 2 个双子叶胞质型（I 和 III） ， 其中双子叶胞质型首先分化出来； 双子叶质体型（II） ，嵌套在双子叶胞质型中； 1 个单子叶胞质型（IV） 和 1 个单子叶质体型（V） 。④根据系统发育树、 蛋白质长度和内含子数目的不同， 将 V 组分为两个支持良好的亚组（V-1 和 V-2） 。 ⑤在 V-1 中， 光合型 NADP-ME 嵌套在质体型非光合型 NADP-ME 中， 两者在内含子数目和蛋白质长度上有相似性。以上研究结果阐明了松叶猪毛菜 NADP-ME 家族基因的特性， 并确定了非生物胁迫下（强光、 干旱和盐） Salchl 的抗逆功能， 同时， 理清了 NADP-ME 家族（特别是对于质体亚型） 在被子植物中的系统发育关系。 |
| Other Abstract | NADP-malic enzyme (NADP-ME) is a key enzyme in malic acid metabolismpathway. It not only participates in plant photosynthesis, but also involves indefensing environmental stress and related metabolism activities. Our previous studiesshowes that the C3-C4 intermediate plant, Salsola laricifolia could resist the droughtstress by enhancing C4 pathway, and only NADP-ME enzymes activity increasesignificantly under different degrees of drought stress, suggesting that this gene mayplay an important role in drought stress. Moreover, the evolutionary relationshipamong NADP-ME isoforms in angiospersm is complex, which provides a goodmaterial to understand the evolution of C4 photosynthetic gene.In this study, we aims to clone the NADP-ME family genes of Salsola laricifola,analyze the expression characteristics of NADP-ME in different tissues and responseto different stress, test the function of NADP-ME under abiotic stress (light, droughtand salt), and clarify the phylogeny and evolution of NADP-ME family inangiosperms. The main results are as follows:1. Based on the transcriptome data, three NADP-MEs are cloned, namely Salchl,Salcyt1, and Salcyt2. ①Their CDS (coding sequence) length are 1941 bp, 1755 bp,1758 bp, encoding 646, 584, 585 amino acids, respectively. All these three genescontain 5 conserved domains of NADP-ME. ② Subcellular localization studies showthat the protein encoded by Salchl is located in chloroplast, and the protein encodedby Salcyt1 and Salcyt2 is located in cytoplasm, respectively. ③On the transcriptionlevel, three genes are all expressed in roots, stems and leaves, but no obvioustissue-specific expression is observed. Under high light, drought and salt stress, thesethree NADP-MEs have different transcription expression patterns in roots and leaves,and only Salchl is significantly induced by these three treatments in roots and leaves.2. The stable transformation of Salchl into Arabidopsis thaliana are used forfunctional verification under abiotic stresses. The results show that: ①under normal conditions, over-expression of Salchl could improve the photosynthetic capacity oftransgenic plants. Compared with WT, the expression of photosynthetic related genes(CHL M, Por B, LHC A2, LHC H4, LHC B3, Psb D, Psb Q, Psa F, and Psa H), ETR(photosynthetic electron transfer rate), Y (II) (actual light energy conversionefficiency) and chlorophyll content are all significantly increased; ②Compared withWT, over-expression of Salchl could not improve the seed germination rate oftransgenic plants under high light, drought and salt stresses; at the seedling stage,over-expression of Salchl can improve the root length, fresh weight and number oflateral roots of transgenic plants in varying degrees under salt, drought and high lightstresses; at the adult stage, over-expression of Salchl decreases the H2O2 content andincreases proline content of transgenic plants under these three stresses. Under saltand strong light stress, the contents of SOD, POD and CAT increase in transgenicplants, but under drought stress, the values of ASA/DHA and GSH/GSSG decrease intransgenic plants. Both ETR and Y(II) of transgenic plants increase under these threestresses.3. Taking advantage of the complete genome sequence recently released, 282 and28 putative NADP-ME genes from 64 species in angiospersm and 10 species inoutgroups, repectively, are used to reassess the phylogeny and evolution of NADP-MEfamily in angiospersm. The results show that, ① NADP-MEs in dicots and monocotsare presented different both in the gene duplication/loss events, motif composition anddistribution. ② All cytosolic NADP-ME sequences exist many differences within theconsensus sequences of each five conserved site. But the plastidic NADP-MEsequences are existed relatively more differences within the consensus sequences inSite II. ③ Four algel sequences are at basal and divergent from the rest of outgroups.These NADP-MEs from angiospersm are classified into five main groups, includingtwo dicot cytosolic groups (I and III), in which Group I dicot cytosolic branched offfirst, one monophyletic dicot plastidic group (II) nested in dicot cytosolic groups, onemonocot cytosolic group (IV) and one monophyletic monocot plastidic group (V). ④Group V is divided into two well-supported subgroups (V-1 and V-2) based on thephylogeny tree, and different range of protein length and intron number. ⑤ In GroupV-1, photosynthetic isoform is nested in plastidic non-photosynthetic isoform, andthey are similar in the range of intron number and protein length. Our study based onextensive sequences from more representative taxa gives new sights into understandthe phylogeny and evolution of NADP-ME family, especially for the plastidic isoform.The results of this study have elucidated the characteristics of NADP-ME familygene of Salsola laricifolia, verified the function of Salchl under abiotic stress (highlight, drought and salt), and clarified the phylogenetic relationship of NADP-MEfamily (especially for plastid subtype) in angiosperms. |
| Subject Area | 植物学 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | http://ir.xjlas.org/handle/365004/15479 |
| Collection | 中国科学院新疆生态与地理研究所 研究系统 |
| Affiliation | 中国科学院新疆生态与地理研究所 |
| First Author Affilication | 中国科学院新疆生态与地理研究所 |
| Recommended Citation GB/T 7714 | 王玉兰. 松叶猪毛菜 NADP-ME 家族基因的克隆及其叶绿体型基因的功能研究[D]. 北京. 中国科学院大学,2020. |
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