‘红叶’杜仲叶片呈色生理特性及关键基因筛选

发布时间：2017-12-28 02:18

本文关键词：‘红叶’杜仲叶片呈色生理特性及关键基因筛选　出处：《中国林业科学研究院》2017年博士论文　论文类型：学位论文

【摘要】：杜仲(Eucommia ulmoides Oliv.)属杜仲科,仅1属1种,是中国重要的名贵经济树种。‘红叶’杜仲(Eucommia ulmoides‘Hongye’)叶片红褐色,极具观赏价值,且药用价值突出,2009年通过河南省林木良种审定。目前对‘红叶’杜仲生理特征及呈色规律研究较少。本论文以‘红叶’杜仲、‘小叶’杜仲(Eucommia ulmoides‘Xiaoye’)以及‘密叶’杜仲(Eucommia ulmoides‘Miye’)三个品种苗期的叶片为研究材料,对其表型性状、微观构造、内部各活性成分含量、光合作用参数及叶绿素荧光参数等指标进行测定,揭示‘红叶’杜仲特殊叶色形成的直接原因;通过转录组测序筛选出控制‘红叶’杜仲叶色形成的关键基因。主要研究结果如下:1.‘红叶’杜仲的叶片正面暗红色,背面绿色,其他品种的叶片正面和背面均为绿色;‘红叶’杜仲的栅栏组织呈现鲜红色,其他品种的栅栏组织为深绿色。‘红叶’杜仲的亮度(L值)为28.17,变红度(a*值)为0.96,变黄度(b*值)为2.69,颜色指数为5.54,达到深红级别。‘红叶’杜仲叶绿素a含量(Ca)为1.10 mg·g-1,叶绿素b含量(Cb)为0.45 mg·g-1,类胡萝卜素含量(CCar)为0.45 mg·g-1,总叶绿素含量(CT)为1.56 mg·g-1,花色苷含量(CA)为0.30 mg·g-1,其CCar、CA以及CCar/CT均显著高于‘密叶’杜仲和‘小叶’杜仲,而‘红叶’杜仲和‘密叶’杜仲间的Ca和Cb及Ca/Cb及CT均无显著差异。高含量的花色苷是‘红叶’杜仲叶片呈现红色的主要原因。2.‘红叶’杜仲叶片最大,其叶长、叶宽及叶面积分别为14.44 cm、6.98 cm和77.34 cm2,显著高于‘小叶’杜仲。‘红叶’杜仲的叶片厚度和栅栏组织厚度分别为431.67μm和186.61μm,其中海绵组织厚度显著低于‘小叶’杜仲,但其栅栏组织厚度则显著高于‘小叶’杜仲。‘红叶’杜仲黄酮质量分数最高,为1.70%,显著高于其他2个品种;绿原酸和京尼平苷酸质量分数分别为1.47%和0.29%,其中绿原酸质量分数与其他2个品种无显著差异,但京尼平苷酸显著低于‘密叶’杜仲。3.‘红叶’杜仲的光补偿点为23.96μmol·m-2·s-1,光饱和点为495.94μmol·m-2·s-1,最大净光合速率为16.91μmol·m-2·s-1,暗呼吸速率1.67μmol·m-2·s-1,初始斜率为0.08;‘小叶’杜仲的光补偿点为26.49μmol·m-2·s-1,光饱和点为521.92μmol·m-2·s-1,最大净光合速率为18.50μmol·m-2·s-1,暗呼吸速率为1.67μmol·m-2·s-1,初始斜率为0.07;‘红叶’杜仲的温度和各光合参数间存在显著的线性回归关系,回归方程的决定系数为0.48~0.96。‘红叶’杜仲的最小荧光(Fo)为0.36,显著低于‘小叶’杜仲,而‘红叶’杜仲Fv/Fm、Fv/Fo及659μmol·m-2·s-1光照条件下的初始荧光(Fo’)分别为0.81,4.18和12.40~12.50;显著高于‘小叶’杜仲;2个品种的光合作用参数和叶绿素荧光参数表明,‘红叶’杜仲利用中强光的能力高于‘小叶’杜仲,而强光的利用能力弱于‘小叶’杜仲。4.‘红叶’杜仲叶色形成过程中,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*值在不同阶段间均存在极显著差异,且其叶色各指标的变化幅度远高于‘小叶’杜仲。在‘红叶’杜仲叶色形成中,其色素含量除了叶绿素a/叶绿素b是逐步降低外,其他指标均为逐步上升,且各指标在叶色形成的不同阶段间均存在极显著差异;花色苷含量从0.22 mg·g-1增至1.91 mg·g-1,增幅达748.96%,增幅远高于‘小叶’杜仲;PAL酶活性在‘红叶’杜仲叶色形成各阶段无显著差异,而CHI酶活性在各阶段存在显著差异。叶片色差值与色素含量直接相关,L值、a*值及b*值与类胡萝卜素含量、花色苷的含量及类黄酮含量成极显著相关;CHI酶活性和杜仲叶片L值、a*值、b*值类胡萝卜素含量、花色苷含量及类黄酮含量间均存在显著或者极显著的相关关系,而PAL酶活性与叶片色差值间则无显著相关性。5.本研究采用RNA-seq技术对‘红叶’杜仲和‘小叶’杜仲的典型叶片进行转录组测序及分析,共得到216,385,416条clean reads,经组装得到54,517条Unigene,组装的N50为1,377 nt,平均长度为806.90 nt,组装效果较好。共有25,993条Unigene得到注释,注释比例为47.68%,其中27,732条基因能编码蛋白序列,占总Unigene数量的50.87%。经转录因子预测,得到5,854个转录因子,其中bHLH为614个,MYB转录因子为569个,NAC为460个,而WD40蛋白仅有60个。54517条Unigenes中共包含17010个完整型SSR位点,占总SSR位点的96.28%。完整型SSR位点共包含67种重复基元,其中出现频率最高的重复基元类型为单核苷酸重复中的A/T(7747个),其次是AG/CT(5039个)和AT/AT(850个)。从花色苷代谢途径、类黄酮代谢途径及类胡萝卜素代谢途径中共找到13个SSR位点。基因差异表达分析得到‘小叶’杜仲与‘红叶’杜仲相比,差异表达的基因有3,904个,其中1,389个上调,2,515个基因下调。其中,差异表达的转录因子628个,其中MYB转录因子26个,bHLH转录因子49个,尚未发现差异表达的WD40转录因子。通过pathway分析得到‘红叶’杜仲和‘小叶’杜仲在花色苷合成途径中差异表达的关键酶基因16条,其中类黄酮糖基转移酶(GT)基因9个,二氢黄酮醇还原酶(DFR)基因5个,黄烷酮-3-羟化酶(F3H)基因1个,类黄酮3,5羟化酶(F3’5’H)基因1个。在花色苷合成途径中差异表达的转录因子有3个,其中2个MYB转录因子,均为调控糖基转运蛋白;1个b HLH转录因子。经荧光定量验证得到参试基因的表达情况与转录组测序所得趋势基本一致,说明转录组测序的可靠性。候选基因的表达模式规律明显,从中选择出叶龄10 d和叶龄34 d是两个基因表达高峰期。基因间协同表达关系较强,认为EuMYB2、EubHLH、EuGT4、EuGT2、EuF3H可能是花色苷合成的关键基因。
[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
【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】：2017
【分类号】：S687

【相似文献】