毛竹光保护及相关基因功能研究

发布时间：2017-12-27 05:29

本文关键词：毛竹光保护及相关基因功能研究　出处：《中国林业科学研究院》2016年博士论文　论文类型：学位论文

【摘要】：强光条件下,植物产生光抑制是一种普遍存在的现象。植物中存在着多种防御机制以防止或减少光抑制,其中叶黄素循环是植物应对光抑制的重要保护机制之一。玉米黄质环氧化酶(ZEP)和紫黄质脱环氧化酶(VDE)是叶黄素循环中的两个关键酶,能够催化叶黄素循环组分紫黄质(V)、玉米黄质(A)和玉米黄质(Z)间的相互转化,其中Z含量增加能够提高植物的光保护能力。本研究以毛竹(Phyllostachys edulis)为材料,在研究了强光条件下毛竹叶片的光抑制特征和叶黄素循环在毛竹叶片光抑制防御中作用的基础上,克隆获得了叶黄素循环关键基因ZEP和VDE同源基因,并对其进行了功能研究。同时也对毛竹Psb S基因及其上游启动子区域进行了初步研究。主要结果如下:1、应用叶绿素荧光技术,研究强光胁迫下毛竹的光抑制特征和叶黄素循环在毛竹叶片强光破坏防御中的作用。结果表明,在夏天中午的强光下或人为强光胁迫下,毛竹叶片最大光化学效率(Fv/Fm)均降低(p0.01),非光化学淬灭(NPQ)均增加;但下午光强减弱或弱光条件下,Fv/Fm可恢复。叶黄素循环抑制剂二硫苏糖醇(DTT)可以抑制紫黄质脱环氧化酶的活性,DTT处理毛竹叶片,抑制其NPQ,使强光下毛竹叶片的Fv/Fm、光化学淬灭(qP)等荧光参数下降幅度明显加大(p0.01),表明叶黄素循环在毛竹叶片光保护机制中具有重要的作用。2、为筛选毛竹光保护相关基因,在基于强光胁迫下毛竹叶片Fv/Fm和NPQ的变化趋势及自然环境中强光照射时间,选择强光胁迫0 h、0.5 h和8 h后3个时间点进行转录组测序。分析结果表明,共计获得了1293个差异表达基因,其中上调基因中包括OHP、SEP、Elip和Psb S等LHC-like基因以及叶黄素循环关键基因ZEP和VDE,这为后续深入研究毛竹光保护相关基因的功能奠定了基础。3、通过RT-PCR和RACE技术,克隆获得了毛竹中编码玉米黄质环氧化酶基因的全长cDNA序列(GenBank No.KP274885),命名为Pe ZEP,并对其进行了功能分析。该基因cDNA全长2532 bp,编码一个含有670个氨基酸残基的多肽。序列分析表明:PeZEP具有ZEP家族的特征结构域,与水稻(Oryza staiva)等禾本科植物的ZEP高度相似(90%以上)。组织特异性表达分析表明,Pe ZEP在根、茎、叶片、叶鞘中均表达,其中叶片中的表达量最高。在毛竹叶片中,强光和干旱能诱导Pe ZEP的表达上调,而4℃低温胁迫和外源ABA则抑制其表达。Pe ZEP在拟南芥中的功能分析显示,过量表达PeZEP增加了转基因拟南芥对强光的敏感性,同时能增加转基因植株的耐旱性。4、利用实时定量PCR技术研究了Pe VDE在不同胁迫条件下的表达情况。结果表明,强光、42℃高温和干旱等胁迫能诱导毛竹叶片中Pe VDE基因表达的增加,而4℃低温胁迫和外源脱落酸(ABA)在短期内诱导Pe VDE的表达,但随着处理时间增加,其表达受到明显抑制。利用拟南芥vde突变体npq1对毛竹编码紫黄质脱环氧化酶基因Pe VDE的功能进行了研究。结果显示,Pe VDE能够异源互补拟南芥突变体npq1,恢复其NPQ能力,且过量表达Pe VDE可以提高拟南芥的NPQ能力,同时减少强光或低温弱光对其造成的光抑制程度。5、在毛竹中Psb S至少存在2个同源基因(Pe Psb S和Pe Psb S1),两者的开放读码框(ORF)分别长810 bp和807 bp,各自编码269和268个氨基酸多肽,具有91.8%的相似性;两者具有相同的基因结构:4个外显子和3个内含子。基因表达分析显示:Pe Psb S和Pe Psb S1具有相似的表达模式,都在毛竹绿色组织中表达,且受强光诱导表达。利用拟南芥突变体npq4对毛竹PsbS基因功能进行了研究,结果显示,Pe Psb S和Pe Psb S1都能够异源互补拟南芥突变体npq4,恢复其NPQ能力,且过量表达Pe Psb S和Pe Psb S1都可以提高拟南芥在强光下的NPQ和Fv/Fm。6、利用与报告基因GUS融合瞬时表达的方法研究了Pe PsbS和Pe PsbS1的上游启动子的活性。利用毛竹基因组数据,设计引物,克隆了Pe Psb S和Pe Psb S1的上游启动子区域,分别位于起始密码子ATG上游2373 bp和1500 bp。进一步序列分析显示,2个基因的启动子区域均存在大量光响应元件。分别将Pe PsbS和Pe PsbS1启动子与报告基因GUS融合,在烟草中瞬时表达,结果表明2个启动子均可启动GUS基因表达,具有生物学活性。本研究为深入了解毛竹的光保护机制奠定了基础,为解释竹子在自然界广泛分布的环境适应性提供了的参考依据,同时也为开发利用毛竹基因资源提供了良好的借鉴。
[Abstract]:Under the condition of strong light, the photoinhibition of plants is a common phenomenon. There are many defense mechanisms in plants to prevent or reduce light inhibition, and the lutein cycle is one of the important protection mechanisms for plants to respond to light inhibition. Zeaxanthin epoxidase (ZEP) and Deepoxidase (VDE) are two key enzymes in the xanthophyll cycle, can catalyze the xanthophyll cycle component of violaxanthin (V) and zeaxanthin (A) and zeaxanthin (Z) mutual conversion between Z, which can increase the content of light protection ability of high plants. In this study, bamboo (Phyllostachys edulis) as material, based on the study of the light bamboo leaves under strong light inhibition characteristics and xanthophyll cycle of photoinhibition in defense role in bamboo leaves on the cloned xanthophyll cycle key genes ZEP and VDE homologous gene, and its function study. At the same time, the Psb S gene and its upstream promoter region were also studied. The main results are as follows: 1. Chlorophyll fluorescence technology was used to study the photoinhibition characteristics of Phyllostachys pubescens under strong light stress and the role of xanthophyll cycle in the protection of Phyllostachys pubescens leaf's strong light damage. The results showed that the maximum photochemical efficiency (Fv/Fm) of Moso bamboo leaves decreased (P0.01), and the non photochemical quenching (NPQ) increased at the summer high noon or under the strong light stress. However, when the light intensity was decreased or the light intensity was low, Fv/Fm could be restored. The xanthophyll cycle inhibitor two dithiothreitol (DTT) can inhibit the Deepoxidase activity, DTT bamboo leaves, the inhibition of NPQ, make the light bamboo leaf Fv/Fm, photochemical quenching (qP) and fluorescence parameters decreased significantly increased (P0.01), indicating that the leaf flavin cycle in leaves of Phyllostachys pubescens the protection mechanism plays an important role. 2, in order to screen related genes of Phyllostachys pubescens photo protection, under the strong light stress, the trend of Fv/Fm and NPQ of Phyllostachys pubescens leaves and the time of strong light exposure in natural environment were selected. Transcriptome sequencing was performed at 3 time points after selecting 0 h, 0.5 h and 8 h under strong light stress. The results showed that a total of 1293 differentially expressed genes were obtained, among which the LHC-like genes including OHP, SEP, Elip and Psb S and the key genes ZEP and VDE of xanthophyll cycle were included in the up regulation genes, which laid the foundation for further research on the functions of Mao Zhuguang protection related genes. 3, through RT-PCR and RACE technology, cloning the full-length cDNA sequence encoding in bamboo Leze (GenBank No.KP274885), named Pe ZEP, and has carried on the analysis. The gene cDNA has a full length of 2532 BP and encodes a polypeptide containing 670 amino acid residues. Sequence analysis showed that PeZEP had the characteristic domain of the ZEP family, which was similar to the ZEP height of rice (Oryza staiva) and other gramineous plants. The expression analysis of tissue specific expression showed that Pe ZEP was expressed in root, stem, leaf and leaf sheath, among which the expression of leaf was the highest. In the leaves of Phyllostachys pubescens, the expression of Pe ZEP was up-regulated by strong light and drought, while low temperature stress at 4 C and exogenous ABA inhibited its expression. Functional analysis of Pe ZEP in Arabidopsis revealed that over expression of PeZEP increased sensitivity of transgenic Arabidopsis to strong light and increased drought tolerance of transgenic plants. 4. The expression of Pe VDE under different stress conditions was studied by real-time quantitative PCR technique. The results showed that the expression of Pe VDE gene in Moso bamboo leaves could be induced by strong light, high temperature and drought at 42 degrees, while the expression of Pe VDE was induced in 4 Pe at low temperature and exogenous abscisic acid (ABA) in a short time, but its expression was inhibited significantly with the increase of treatment time. The function of Phyllostachys pubescens encoding purplish yellow decycloid oxidase gene Pe VDE was studied using Arabidopsis VDE mutant npq1. The results showed that Pe VDE could complement Arabidopsis mutant npq1 and restore its NPQ ability, and overexpression of Pe VDE could enhance the NPQ ability of Arabidopsis thaliana, and reduce the degree of photoinhibition caused by strong light or low temperature and low light. In 5, Psb S in bamboo has at least 2 homologous genes (Pe and Psb S Pe Psb S1), the open reading frame (ORF) were 810 BP and 807 BP, respectively encoding 269 and 268 amino acid peptide, has 91.8% similarity; both have the same gene structure: 4 exons and 3 introns. Gene expression analysis showed that Pe Psb S and Pe Psb S1 had similar expression patterns, all expressed in green tissues of Phyllostachys pubescens, and were induced by strong light. The function of PsbS gene on bamboo was studied, using Arabidopsis mutant npq4 showed that Pe Psb S and Pe Psb S1 are capable of complementation of Arabidopsis mutant npq4, recovery ability of NPQ, and overexpression of Pe Psb S and Pe Psb S1 can improve the Arabidopsis under strong light NPQ and Fv /Fm. 6. The activity of the upstream promoter of Pe PsbS and Pe PsbS1 was studied by the method of transient expression with the reportable gene GUS. Primers were designed by using genomic data of Phyllostachys pubescens, and the upstream promoter regions of Pe Psb S and Pe Psb S1 were cloned, which were located at the upstream of the start codon ATG 2373 BP and 1500 BP respectively. Further sequence analysis showed that there were a large number of light response elements in the promoter regions of the 2 genes. The Pe PsbS and Pe PsbS1 promoters were fused with reporter gene GUS and transiently expressed in tobacco. The results showed that all 2 promoters could initiate GUS gene expression and had biological activity. This study laid a foundation for further understanding of the light protection mechanism of Moso bamboo. It provided a reference for explaining the environmental adaptability of bamboos widely distributed in nature, and also provided a good reference for the development and utilization of genetic resources of Moso bamboo.
【学位授予单位】：中国林业科学研究院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S795.7

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