本文關鍵詞：‘紅葉’杜仲葉片呈色生理特性及關鍵基因篩選　出處：《中國林業(yè)科學研究院》2017年博士論文　論文類型：學位論文

  更多相關文章： ‘紅葉’杜仲 花色苷 呈色組織定位 光合作用 轉錄組

【摘要】：杜仲(Eucommia ulmoides Oliv.)屬杜仲科,僅1屬1種,是中國重要的名貴經(jīng)濟樹種�！t葉’杜仲(Eucommia ulmoides‘Hongye’)葉片紅褐色,極具觀賞價值,且藥用價值突出,2009年通過河南省林木良種審定。目前對‘紅葉’杜仲生理特征及呈色規(guī)律研究較少。本論文以‘紅葉’杜仲、‘小葉’杜仲(Eucommia ulmoides‘Xiaoye’)以及‘密葉’杜仲(Eucommia ulmoides‘Miye’)三個品種苗期的葉片為研究材料,對其表型性狀、微觀構造、內部各活性成分含量、光合作用參數(shù)及葉綠素熒光參數(shù)等指標進行測定,揭示‘紅葉’杜仲特殊葉色形成的直接原因;通過轉錄組測序篩選出控制‘紅葉’杜仲葉色形成的關鍵基因。主要研究結果如下:1.‘紅葉’杜仲的葉片正面暗紅色,背面綠色,其他品種的葉片正面和背面均為綠色;‘紅葉’杜仲的柵欄組織呈現(xiàn)鮮紅色,其他品種的柵欄組織為深綠色。‘紅葉’杜仲的亮度(L值)為28.17,變紅度(a*值)為0.96,變黃度(b*值)為2.69,顏色指數(shù)為5.54,達到深紅級別。‘紅葉’杜仲葉綠素a含量(Ca)為1.10 mg·g-1,葉綠素b含量(Cb)為0.45 mg·g-1,類胡蘿卜素含量(CCar)為0.45 mg·g-1,總葉綠素含量(CT)為1.56 mg·g-1,花色苷含量(CA)為0.30 mg·g-1,其CCar、CA以及CCar/CT均顯著高于‘密葉’杜仲和‘小葉’杜仲,而‘紅葉’杜仲和‘密葉’杜仲間的Ca和Cb及Ca/Cb及CT均無顯著差異。高含量的花色苷是‘紅葉’杜仲葉片呈現(xiàn)紅色的主要原因。2.‘紅葉’杜仲葉片最大,其葉長、葉寬及葉面積分別為14.44 cm、6.98 cm和77.34 cm2,顯著高于‘小葉’杜仲�！t葉’杜仲的葉片厚度和柵欄組織厚度分別為431.67μm和186.61μm,其中海綿組織厚度顯著低于‘小葉’杜仲,但其柵欄組織厚度則顯著高于‘小葉’杜仲�！t葉’杜仲黃酮質量分數(shù)最高,為1.70%,顯著高于其他2個品種;綠原酸和京尼平苷酸質量分數(shù)分別為1.47%和0.29%,其中綠原酸質量分數(shù)與其他2個品種無顯著差異,但京尼平苷酸顯著低于‘密葉’杜仲。3.‘紅葉’杜仲的光補償點為23.96μmol·m-2·s-1,光飽和點為495.94μmol·m-2·s-1,最大凈光合速率為16.91μmol·m-2·s-1,暗呼吸速率1.67μmol·m-2·s-1,初始斜率為0.08;‘小葉’杜仲的光補償點為26.49μmol·m-2·s-1,光飽和點為521.92μmol·m-2·s-1,最大凈光合速率為18.50μmol·m-2·s-1,暗呼吸速率為1.67μmol·m-2·s-1,初始斜率為0.07;‘紅葉’杜仲的溫度和各光合參數(shù)間存在顯著的線性回歸關系,回歸方程的決定系數(shù)為0.48~0.96。‘紅葉’杜仲的最小熒光(Fo)為0.36,顯著低于‘小葉’杜仲,而‘紅葉’杜仲Fv/Fm、Fv/Fo及659μmol·m-2·s-1光照條件下的初始熒光(Fo’)分別為0.81,4.18和12.40~12.50;顯著高于‘小葉’杜仲;2個品種的光合作用參數(shù)和葉綠素熒光參數(shù)表明,‘紅葉’杜仲利用中強光的能力高于‘小葉’杜仲,而強光的利用能力弱于‘小葉’杜仲。4.‘紅葉’杜仲葉色形成過程中,其L值由32.89降至28.45,a*值由-8.70升至1.03,b*值由13.35降至2.51,L值、a*值及b*值在不同階段間均存在極顯著差異,且其葉色各指標的變化幅度遠高于‘小葉’杜仲。在‘紅葉’杜仲葉色形成中,其色素含量除了葉綠素a/葉綠素b是逐步降低外,其他指標均為逐步上升,且各指標在葉色形成的不同階段間均存在極顯著差異;花色苷含量從0.22 mg·g-1增至1.91 mg·g-1,增幅達748.96%,增幅遠高于‘小葉’杜仲;PAL酶活性在‘紅葉’杜仲葉色形成各階段無顯著差異,而CHI酶活性在各階段存在顯著差異。葉片色差值與色素含量直接相關,L值、a*值及b*值與類胡蘿卜素含量、花色苷的含量及類黃酮含量成極顯著相關;CHI酶活性和杜仲葉片L值、a*值、b*值類胡蘿卜素含量、花色苷含量及類黃酮含量間均存在顯著或者極顯著的相關關系,而PAL酶活性與葉片色差值間則無顯著相關性。5.本研究采用RNA-seq技術對‘紅葉’杜仲和‘小葉’杜仲的典型葉片進行轉錄組測序及分析,共得到216,385,416條clean reads,經(jīng)組裝得到54,517條Unigene,組裝的N50為1,377 nt,平均長度為806.90 nt,組裝效果較好。共有25,993條Unigene得到注釋,注釋比例為47.68%,其中27,732條基因能編碼蛋白序列,占總Unigene數(shù)量的50.87%。經(jīng)轉錄因子預測,得到5,854個轉錄因子,其中bHLH為614個,MYB轉錄因子為569個,NAC為460個,而WD40蛋白僅有60個。54517條Unigenes中共包含17010個完整型SSR位點,占總SSR位點的96.28%。完整型SSR位點共包含67種重復基元,其中出現(xiàn)頻率最高的重復基元類型為單核苷酸重復中的A/T(7747個),其次是AG/CT(5039個)和AT/AT(850個)。從花色苷代謝途徑、類黃酮代謝途徑及類胡蘿卜素代謝途徑中共找到13個SSR位點�；虿町惐磉_分析得到‘小葉’杜仲與‘紅葉’杜仲相比,差異表達的基因有3,904個,其中1,389個上調,2,515個基因下調。其中,差異表達的轉錄因子628個,其中MYB轉錄因子26個,bHLH轉錄因子49個,尚未發(fā)現(xiàn)差異表達的WD40轉錄因子。通過pathway分析得到‘紅葉’杜仲和‘小葉’杜仲在花色苷合成途徑中差異表達的關鍵酶基因16條,其中類黃酮糖基轉移酶(GT)基因9個,二氫黃酮醇還原酶(DFR)基因5個,黃烷酮-3-羥化酶(F3H)基因1個,類黃酮3,5羥化酶(F3’5’H)基因1個。在花色苷合成途徑中差異表達的轉錄因子有3個,其中2個MYB轉錄因子,均為調控糖基轉運蛋白;1個b HLH轉錄因子。經(jīng)熒光定量驗證得到參試基因的表達情況與轉錄組測序所得趨勢基本一致,說明轉錄組測序的可靠性。候選基因的表達模式規(guī)律明顯,從中選擇出葉齡10 d和葉齡34 d是兩個基因表達高峰期。基因間協(xié)同表達關系較強,認為EuMYB2、EubHLH、EuGT4、EuGT2、EuF3H可能是花色苷合成的關鍵基因。
[Abstract]:(Eucommia ulmoides Oliv.) is Eucommia Eucommiaceae, only 1 genera and 1 species, is an important Chinese valuable economic tree species. "Red leaf" Eucommia (Eucommia ulmoides' Hongye ") leaves reddish brown, highly ornamental value, and the medicinal value of outstanding, in 2009 through the forest of Henan Province approved. At present, there are few studies on the physiological characteristics and color rule of Eucommia ulmoides "red leaf". This paper takes "red" and "leaflets" (Eucommia ulmoides Eucommia ulmoides' Xiaoye ') and' dense leaf of Eucommia ulmoides (Eucommia ulmoides "" Miye ") of three varieties of seedling leaves as the research material, the phenotypic traits, microscopic structure, the active ingredient content, photosynthetic parameters and chlorophyll fluorescence parameters" the direct cause was determined, revealing the red leaves' Eucommia special leaf color formed by transcriptome sequencing; selected control "key genes of Eucommia ulmoides leaf color of leaves.". The main results were as follows: 1., the leaves of Eucommia ulmoides leaves were dark red and greenish on the back, and the leaves on the front and back of both varieties were green. The palisade tissues of red leaf Eucommia ulmoides were bright red, and the palisade tissues of other varieties were dark green. The brightness (L value) of "red leaf" Eucommia ulmoides is 28.17, the redness (a*) is 0.96, the yellowing (b*) is 2.69 and the color index is 5.54, reaching the deep red grade. "Red" Eucommia chlorophyll a (Ca) 1.10 Mg - g-1, the content of chlorophyll b (Cb) 0.45 mg - g-1, carotenoid content (CCar) was 0.45 mg - g-1, the total chlorophyll content (CT) was 1.56 Mg - g-1, anthocyanin content (CA) was 0.30 Mg - g-1 the CCar, CA, and CCar/CT were significantly higher than that of "dense leaf" and "leaflets" Eucommia eucommia, "but there were no significant differences between the leaves of Eucommia ulmoides' and 'dense leaf' between Ca and Cb and eucommia Ca/Cb and CT. High content anthocyanins are the main reasons for the red leaves of "red leaf" Eucommia ulmoides. The leaves of 2. 'red leaf' Eucommia were the largest, the leaf length, Ye Kuan and leaf area were 14.44 cm, 6.98 cm and 77.34 cm2 respectively, which were significantly higher than those of the 'lobule' Eucommia. The leaf thickness and palisade tissue thickness of 'Hongye' Eucommia ulmoides were 431.67 and 186.61 m, respectively. The thickness of sponge tissue was significantly lower than that of 'lobular' Eucommia ulmoides, but the palisade tissue thickness of M was significantly higher than that of 'lobular' Eucommia ulmoides. The 'Red' quality of Eucommia flavonoids highest score was 1.70%, significantly higher than the other 2 varieties; chlorogenic acid and geniposidic acid content was 1.47% and 0.29% respectively, which had no significant difference in the content of chlorogenic acid and other 2 varieties, but geniposidic acid was significantly lower than that of "dense leaf Eucommia". The light compensation point of 3. "red" Eucommia is 23.96 mol - m-2 - s-1, the light saturation point is 495.94 mol - m-2 - s-1, the maximum net photosynthetic rate of 16.91 mol - m-2 - s-1, the dark respiration rate 1.67 mol - m-2 - s-1, the initial slope is 0.08; the light compensation point "the 26.49 leaflets of Eucommia ulmoides mol - m-2 - s-1, the light saturation point is 521.92 mol - m-2 - s-1, the maximum net photosynthetic rate of 18.50 mol - m-2 - s-1, the respiration rate of 1.67 mol - m-2 - s-1, the initial slope of 0.07;" red "and the temperature of Eucommia ulmoides photosynthetic parameters have significant linear relationship, the coefficient of the regression equation for 0.48~0.96 decision. "The minimum fluorescence of Eucommia ulmoides leaves" (Fo) was 0.36, significantly lower than the "lobular 'eucommia, and' Red 'Fv/Fm, Eucommia Fv/Fo and 659 Mu mol - m-2 - S-1 light under the condition of the initial fluorescence (Fo) were 0.81,4.18 and 12.40~12.50 were significantly higher than that of" lobular "; Eucommia ulmoides; photosynthetic parameters and show the chlorophyll fluorescence parameters of 2 varieties," red "Du nakatoshi ability light is higher than that in" lobular "eucommia, and the ability to use light less than" leaflets "of Eucommia ulmoides. 4., the L value of red leaves of Eucommia ulmoides leaves was reduced from 32.89 to 28.45, a* value increased from -8.70 to 1.03, b* value decreased from 13.35 to 2.51, L value, a* value and b* value had significant difference at different stages, and their leaf color indexes were much higher than those of 'little leaf' Eucommia ulmoides. In the "red" color in the form of eucommia leaf pigment content, in addition to chlorophyll a/ chlorophyll b is gradually reduced, other indexes were gradually increased, was significantly different in different stages and each index is formed in the leaf color; anthocyanin content from 0.22 mg g-1 to 1.91 Mg - g-1, an increase of 748.96% that increase is much higher than the "lobular" Eucommia; PAL activity in 'Red' eucommia leaf color formation in each stage had no significant difference, but significant differences of CHI activity in each stage. Leaf color value is directly related to the pigment content, L value, a* value and b* value and the content of carotenoid, anthocyanin and flavonoid content was highly correlated; CHI enzyme activity and eucommia leaf L value, a* value, b* value of carotenoid content, anthocyanin content and flavonoid content were significant or very significant correlation, while PAL activity and leaf color value had no significant relationship. 5. this study uses RNA-seq technology transcriptome sequencing and analysis of typical "red" and "leaflets" of Eucommia eucommia, there were 216385416 clean reads and 54517 Unigene are obtained by the assembly, the assembly of the N50 is 1377 NT, the average length of 806.90 NT, the assembly effect is good. A total of 25993 Unigene were annotated with an annotation ratio of 47.68%, of which 27732 genes encoded a protein sequence, accounting for 50.87% of the total number of Unigene. Through the prediction of transcription factors, 5854 transcription factors were obtained, of which 614 were bHLH, 569 for MYB transcription factors, 460 for NAC, and only 60 for WD40 protein. 54517 Unigenes included 17010 complete SSR loci, accounting for 96.28% of the total SSR site. The complete SSR loci contained 67 repeated primitives. Among them, the highest repetition primitives were A/T (7747), followed by AG/CT (5039) and AT/AT (850). 13 SSR loci were found from anthocyanin metabolism pathway, flavonoid metabolism pathway and carotenoid metabolism pathway. gene
【學位授予單位】：中國林業(yè)科學研究院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S687

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7 王淋;杜仲基因組分析及適應性和橡膠生物合成的分子基礎研究[D];中國林業(yè)科學研究院;2017年

8 杜紅巖;杜仲含膠特性及其變異規(guī)律與無性系選擇的研究[D];中南林學院;2003年

9 王大瑋;杜仲遺傳連鎖圖譜構建及重要性狀的分子標記[D];西北農(nóng)林科技大學;2011年

10 劉世會;杜仲蛋白純化、序列分析及抗菌活性研究[D];貴州大學;2008年

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1 何文廣;杜仲不同器官杜仲膠含量、相對分子質量及其分布的動態(tài)研究[D];西北農(nóng)林科技大學;2009年

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10 陳洋;杜仲雄花粉蛋白成分的分離純化研究[D];河南大學;2015年

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