本文關(guān)鍵詞：毛竹光保護及相關(guān)基因功能研究　出處：《中國林業(yè)科學(xué)研究院》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 毛竹 光保護 葉黃素循環(huán) 玉米黃質(zhì)環(huán)氧化酶 紫黃質(zhì)脫環(huán)氧化酶

【摘要】：強光條件下,植物產(chǎn)生光抑制是一種普遍存在的現(xiàn)象。植物中存在著多種防御機制以防止或減少光抑制,其中葉黃素循環(huán)是植物應(yīng)對光抑制的重要保護機制之一。玉米黃質(zhì)環(huán)氧化酶(ZEP)和紫黃質(zhì)脫環(huán)氧化酶(VDE)是葉黃素循環(huán)中的兩個關(guān)鍵酶,能夠催化葉黃素循環(huán)組分紫黃質(zhì)(V)、玉米黃質(zhì)(A)和玉米黃質(zhì)(Z)間的相互轉(zhuǎn)化,其中Z含量增加能夠提高植物的光保護能力。本研究以毛竹(Phyllostachys edulis)為材料,在研究了強光條件下毛竹葉片的光抑制特征和葉黃素循環(huán)在毛竹葉片光抑制防御中作用的基礎(chǔ)上,克隆獲得了葉黃素循環(huán)關(guān)鍵基因ZEP和VDE同源基因,并對其進行了功能研究。同時也對毛竹Psb S基因及其上游啟動子區(qū)域進行了初步研究。主要結(jié)果如下:1、應(yīng)用葉綠素?zé)晒饧夹g(shù),研究強光脅迫下毛竹的光抑制特征和葉黃素循環(huán)在毛竹葉片強光破壞防御中的作用。結(jié)果表明,在夏天中午的強光下或人為強光脅迫下,毛竹葉片最大光化學(xué)效率(Fv/Fm)均降低(p0.01),非光化學(xué)淬滅(NPQ)均增加;但下午光強減弱或弱光條件下,Fv/Fm可恢復(fù)。葉黃素循環(huán)抑制劑二硫蘇糖醇(DTT)可以抑制紫黃質(zhì)脫環(huán)氧化酶的活性,DTT處理毛竹葉片,抑制其NPQ,使強光下毛竹葉片的Fv/Fm、光化學(xué)淬滅(qP)等熒光參數(shù)下降幅度明顯加大(p0.01),表明葉黃素循環(huán)在毛竹葉片光保護機制中具有重要的作用。2、為篩選毛竹光保護相關(guān)基因,在基于強光脅迫下毛竹葉片F(xiàn)v/Fm和NPQ的變化趨勢及自然環(huán)境中強光照射時間,選擇強光脅迫0 h、0.5 h和8 h后3個時間點進行轉(zhuǎn)錄組測序。分析結(jié)果表明,共計獲得了1293個差異表達基因,其中上調(diào)基因中包括OHP、SEP、Elip和Psb S等LHC-like基因以及葉黃素循環(huán)關(guān)鍵基因ZEP和VDE,這為后續(xù)深入研究毛竹光保護相關(guān)基因的功能奠定了基礎(chǔ)。3、通過RT-PCR和RACE技術(shù),克隆獲得了毛竹中編碼玉米黃質(zhì)環(huán)氧化酶基因的全長cDNA序列(GenBank No.KP274885),命名為Pe ZEP,并對其進行了功能分析。該基因cDNA全長2532 bp,編碼一個含有670個氨基酸殘基的多肽。序列分析表明:PeZEP具有ZEP家族的特征結(jié)構(gòu)域,與水稻(Oryza staiva)等禾本科植物的ZEP高度相似(90%以上)。組織特異性表達分析表明,Pe ZEP在根、莖、葉片、葉鞘中均表達,其中葉片中的表達量最高。在毛竹葉片中,強光和干旱能誘導(dǎo)Pe ZEP的表達上調(diào),而4℃低溫脅迫和外源ABA則抑制其表達。Pe ZEP在擬南芥中的功能分析顯示,過量表達PeZEP增加了轉(zhuǎn)基因擬南芥對強光的敏感性,同時能增加轉(zhuǎn)基因植株的耐旱性。4、利用實時定量PCR技術(shù)研究了Pe VDE在不同脅迫條件下的表達情況。結(jié)果表明,強光、42℃高溫和干旱等脅迫能誘導(dǎo)毛竹葉片中Pe VDE基因表達的增加,而4℃低溫脅迫和外源脫落酸(ABA)在短期內(nèi)誘導(dǎo)Pe VDE的表達,但隨著處理時間增加,其表達受到明顯抑制。利用擬南芥vde突變體npq1對毛竹編碼紫黃質(zhì)脫環(huán)氧化酶基因Pe VDE的功能進行了研究。結(jié)果顯示,Pe VDE能夠異源互補擬南芥突變體npq1,恢復(fù)其NPQ能力,且過量表達Pe VDE可以提高擬南芥的NPQ能力,同時減少強光或低溫弱光對其造成的光抑制程度。5、在毛竹中Psb S至少存在2個同源基因(Pe Psb S和Pe Psb S1),兩者的開放讀碼框(ORF)分別長810 bp和807 bp,各自編碼269和268個氨基酸多肽,具有91.8%的相似性;兩者具有相同的基因結(jié)構(gòu):4個外顯子和3個內(nèi)含子。基因表達分析顯示:Pe Psb S和Pe Psb S1具有相似的表達模式,都在毛竹綠色組織中表達,且受強光誘導(dǎo)表達。利用擬南芥突變體npq4對毛竹PsbS基因功能進行了研究,結(jié)果顯示,Pe Psb S和Pe Psb S1都能夠異源互補擬南芥突變體npq4,恢復(fù)其NPQ能力,且過量表達Pe Psb S和Pe Psb S1都可以提高擬南芥在強光下的NPQ和Fv/Fm。6、利用與報告基因GUS融合瞬時表達的方法研究了Pe PsbS和Pe PsbS1的上游啟動子的活性。利用毛竹基因組數(shù)據(jù),設(shè)計引物,克隆了Pe Psb S和Pe Psb S1的上游啟動子區(qū)域,分別位于起始密碼子ATG上游2373 bp和1500 bp。進一步序列分析顯示,2個基因的啟動子區(qū)域均存在大量光響應(yīng)元件。分別將Pe PsbS和Pe PsbS1啟動子與報告基因GUS融合,在煙草中瞬時表達,結(jié)果表明2個啟動子均可啟動GUS基因表達,具有生物學(xué)活性。本研究為深入了解毛竹的光保護機制奠定了基礎(chǔ),為解釋竹子在自然界廣泛分布的環(huán)境適應(yīng)性提供了的參考依據(jù),同時也為開發(fā)利用毛竹基因資源提供了良好的借鑒。
[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.
【學(xué)位授予單位】：中國林業(yè)科學(xué)研究院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S795.7

【參考文獻】

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