【作者】 杨念婉；

【导师】 李艾莲；

【作者基本信息】 中国协和医科大学 ， 生药学， 2010， 博士

【摘要】 薏苡(Coix lachryrma-jobi L.),是我国古老的药粮兼用作物,属于禾本科(Gramineae),玉米族(Tripasacea),薏苡属(Coix),为一年生或多年生的C4草本植物。薏苡仁收载于中国药典2000年版一部,具健脾渗湿,除痹止泻,清热排脓之功效,中医用于治疗水肿、小便不利、脾虚泄泻、肺痈、肠痈、扁平疣。近年来中外学者陆续报道了薏苡抗肿瘤、免疫调节、降血糖、降压、抗病毒等方面的药理活性。此外,薏苡营养价值高并且全面,享有“世界禾本科植物之王”的美誉。我国薏苡资源丰富,但生产上缺乏优质薏苡新品种,现有品种普遍存在产量较低,植株过高易倒伏、熟期偏迟、抗病虫性差等问题,很大程度上制约了薏苡生产的发展。本研究以中国医学科学院协和医学院药用植物研究所经神州一号航天搭载[(99)京证经字66390号]的薏苡(Coix lachryrma-jobi L.)经系统选育的2个品系一一协和薏苡Ⅰ号、协和薏苡Ⅱ号为研究材料,比较二者的表型、生化和细胞遗传学差异,分析薏苡产量形成的主导因子,并通过不同养分、密度和播期对产量和品质进行调控,从而一方面选择优良的薏苡品系以利于日后推广,另一方面为指导薏苡的遗传育种提供有益信息。研究结果概括如下：1.表观形态特征、同功酶及细胞遗传学研究表观形态上,2个薏苡品系差异主要表现为协和薏苡Ⅰ号的幼嫩叶片颜色、茎色以及柱头颜色均为紫色,而协和薏苡Ⅱ号均为绿色。同功酶酶谱分析结果表明,2个薏苡品系的过氧化物酶(POD)和超氧化物歧化酶(SOD)酶谱基本相同,差异表现在,相对于协和薏苡Ⅱ号,协和薏苡Ⅰ号的POD同功酶谱带数目较多,酶活性较强,说明2个品系存在一定的遗传差异。细胞染色体核型分析结果表明,2个薏苡品系的核型公式均为2n=20=16m+4sm,染色体核型对称性为1A型；二者在染色体形态上存在一定的差异：Ⅰ号和Ⅱ号薏苡的染色体绝对长度分别为3.01-5.36um和2.25-7.99 gm,染色体长度比分别为1.78和3.55,说明二者在遗传性状上有一定的差异。2.药用和食用品质研究对薏苡仁的药典标定成分——甘油三油酸酯的含量分析以及不同养分、播期对其的影响研究表明,Ⅰ号(1.17%)和Ⅱ号(0.97%)薏苡仁甘油三油酸酯含量均高于《中华人民共和国药典2010版(一部)》所要求的0.4%。磷肥、钾肥配施可显著提高Ⅰ号(70.0%)和Ⅱ号(78.2%)薏苡仁甘油三油酸酯含量。播期从4月27日推迟到5月17日薏苡仁甘油三油酸酯含量增加(Ⅰ号27.2%；Ⅱ号22.8%)。薏苡仁的营养成分研究表明,Ⅰ号和Ⅱ号薏苡的蛋白质含量分别为16.7%和16.5%,脂肪为5.6%和6.6%。2种薏苡仁的谷氨酸(Glu)、亮氨酸(Leu)含量丰富(平均含量分别为39.1 mg/g、24.1 mg/g)；色氨酸(Trp)、赖氨酸(Lys)含量较低；但含硫氨基酸(Met+Cys)含量丰富。以氨基酸评分(AAS)为标准,2种薏苡仁的第一限制性氨基酸均为色氨酸(Trp),第二限制性氨基酸为赖氨酸(Lys)。Ⅰ号和Ⅱ号薏苡仁的氨基酸与总氨基酸比值(EAA/TAA)分别为37.2%和37.6%、必需氨基酸与非必需氨基酸比值(EAA/NEAA)分别为0.59和0.60、评价必需氨基酸指数(EAAI)分别为47.71和48.55。Ⅰ号薏苡仁的油酸、亚油酸和棕榈酸含量分别为32.2、20.5和6.8 mg/g,均略高于Ⅱ号薏苡。3.产量构成因素分析与内源激素调节研究与现有研究的薏苡品种相比,本研究中的协和薏苡Ⅰ号在产量上有很大幅度的提高。产量构成因素相关性分析和通径分析表明,地上部生物产量、单株穗数、分蘖数是薏苡产量的重要构成因子。主成分分析表明,协和薏苡Ⅰ号单株穗数多、生物产量高、分蘖数多,且穗着粒数和百粒重适中,种质优良：协和薏苡Ⅱ号单株穗数少、生物产量低、分蘖数少、穗着粒数少,百粒重适中。分蘖数是薏苡产量的重要构成因素,禾本科植物分蘖的发生与内源激素密切相关。对2个薏苡品系在分蘖发生过程中的内源激素变化的研究结果表明,较高的生长素吲哚乙酸(IAA)含量对分蘖发生不利,而较高的细胞分裂素玉米素核苷(ZR)含量则有利于分蘖的发生,脱落酸(ABA)对薏苡分蘖的作用不大。IAA和ZR以互作的方式参与分蘖发生过程,在整个分蘖发生过程中,分蘖数较多的Ⅰ号薏苡的IAA/ZR比值始终低于Ⅱ号薏苡,并且在分蘖开始和结束的时段差异最为明显,同时,Ⅰ号薏苡在此两个阶段分蘖的增长速度也显著快于Ⅱ号薏苡。4.光合荧光特性研究薏苡光合色素含量测定表明,苗期和生长期,Ⅰ号薏苡的叶绿素a含量低于Ⅱ号薏苡,但类胡萝卜素含量高于Ⅱ号薏苡；Ⅰ号薏苡通过提高叶绿素b的含量来降低叶绿素a、b的比值,从而增加叶片对光能的捕获；而在成熟期,Ⅰ号慧苡的叶绿素a含量显著升高。薏苡的净光合速率日变化呈单峰曲线,上午随着光合有效辐射的增加,净光合速率显著升高,Ⅰ号薏苡升高的速度快于Ⅱ号薏苡；两者均在12：00时达到最大值,其中,Ⅰ号薏苡净光合速率达到18.2μmol CO2m-2s-1,Ⅱ号薏苡净光合速率为18.9μmolC02 m-2s-1；以后逐渐下降,日落时的净光合速率低于清晨。光响应曲线研究结果表明,Ⅰ号薏苡的光补偿点(LCP)较低(24.0μmol m -2S-1),光饱和点(LSP)较高(1197μmol m-2s-1),对光强的利用范围比Ⅱ号薏苡(LCP为50.0μmol m-2s-1, LSP为931μmol m-2s-1)广。叶绿素荧光测定结果表明,Ⅰ号薏苡的Fo、Fv/Fm、ΦPSⅡ和qP均高于Ⅱ号薏苡,表明Ⅰ号薏苡植株叶片的PSⅡ反应中心的开放程度较高,具有较高的电子传递活性和光能转换效率；而Ⅱ号薏苡的NPQ较高,表明其PSⅡ的潜在热耗散能力较强,可有效地避免过剩光能对光合机构的损伤。5.抗氧化酶活性测定苗期,Ⅱ号薏苡的超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)活性均显著高于Ⅰ号薏苡,过氧化物酶(POD)、过氧化氢酶(CAT)、谷胱甘肽还原酶(GR)以及花青素和类黄酮均显著低于Ⅰ号薏苡；生长期,Ⅱ号薏苡的SOD、POD、APX活性均显著高于Ⅰ号薏苡,CAT以及花青素和类黄酮均显著低于Ⅰ号薏苡；开花期,Ⅱ号薏苡的SOD、POD、APX活性均显著高于Ⅰ号薏苡,GR以及花青素和类黄酮均显著低于Ⅰ号薏苡。Ⅱ号薏苡对抗自由基主要通过抗氧化酶系的作用,Ⅰ号薏苡则具有花青素和类黄酮含量上的优势。6.养分、密度和播期对产量性能的调控研究不同养分施用水平和组合下,薏苡产量及其构成因素的差异达显著或极显著水平。Ⅰ号薏苡N1P2K2处理的产量最高,达到284.4 kg/667m2,比无养分处理产量增加136.8%，产值高达2491元/667m2。通过模型估算Ⅰ号薏苡的经济最佳纯氮、纯磷和纯钾的施用量为6.64、5.38、6.63 kg/667m2。Ⅰ号薏苡产量的增加主要通过提高粒数和结实率,故磷和钾是提高其产量的重要养分,养分吸收结果分析表明可以适当降低氮肥的施用量来增加Ⅰ号薏苡的磷、钾吸收。不同种植密度主要通过影响薏苡分蘖数的多少对薏苡产量产生显著影响,稀植2700株/667m2(株行距60cm×80cm)的经济效益最高,达2132元/667m2,且有利于减少病虫的为害。播期推迟20天,Ⅰ号薏苡的产量降低8.2%，各产量构成因素中除了分蘖数减少外,其它各因素受播期影响不大；考虑播期提前人力、灌溉等消耗增加,而薏苡仁药用成分甘油三油酸酯含量减少,建议在北京地区种植适宜播期为5月中旬。更多还原

【Abstract】 Coix (Coix lachryrma-jobi L.), an erect annual or perennial herb of Gramineae, is used as Chinese traditional folk medicine for treatment of edema, dysuria, diarrhea of spleen deficiency, lung carbuncle, periappendicular abscess and verruca plana. It has been contained in Chinese Pharmacopoeia since 2000. The pharmacological activities of anti-tumor, immunoregulation, hypoglycemic, blood pressure decreasing and anti-virus have been reported in rensent years. In addition, coix was titled as "King of the world Gramineas" since the high nutrition values.The resources of coix in China are rich, but the problem of low yield, laid plant, late mature period and the poor ability of desease and insect resistance constrainted the development of coix production. For further selection of good variety to facilitate the future promotion provide useful information of genetic breeding of coix, the main factors affecting the yield and quality information of the two stains of coix which breed and selected from space mutation were researched in this study. The results were as follows:1. Morphology, isozyme and cytogenetis studyThe differences of 2 coix stains in plant morphology were presented on the color of young leaves, stem and stigma. The colors of these parts of Xiehe CoixⅠwere purple, while the Xiehe CoixⅡwere green.In the analysis of isozyme pattens of 2 coix stains, it was found that the SOD and POD isozymes of these 2 stains were basically the same, which indicated a close genetic relationship between them. The POD isozymes of Xiehe Coix I had a lager number and stronger activity, as compared with Xiehe CoixⅡ, which indicate that there were some genetic differences between these 2 stains, and they may belong to the different physical types of the same species.The karyotype formula of the 2 coix stains was 2n= 20= 16m+4sm and the karyotype symmetry was 1A-type. The absolute length of chromosomes of Xiehe CoixⅠand Xie CoixⅡwere 3.01-5.36μm and 2.25-7.99μm, respectively, while the chromosome length ratios were 1.78 and 3.55, respectively. The differences in chromosome morphology of these 2 stains indicated that space mutation had caused different degrees of variation.2. Evaluation the main qualities of coixThe contents of triolein in Xiehe CoixⅠ(1.17%) and Xiehe CoixⅡ(0.97%) were higher than the requirement of "The People’s Republic of China Pharmacopoeia 2005 edition (1)". The delay of planting date caused the increasing of triolein content of coicis. The application of potassium and phosphate fertilizers can raised the content of triolein in Xiehe CoixⅠ(70.0%) and Xiehe CoixⅡ(78.2%) significantly, while the application of nitrogen fertilizer had no effect on triolein content.The contents of protein of Xiehe CoixⅠand Xiehe CoixⅡwere 16.7% and 16.5%, respectively, whihe the contents of fat were 5.6% and 6.6%, respectively. The contents of Glu and Leu of the 2 coix were quite high, while Trp and Lys were low. According to ASS, Trp was the first limiting amino acid, and the second limiting amino acid was Lys. The EAA/TAA of Xiehe CoixⅠand Xie CoixⅡwas 37.2% and 37.6%, respectively, while the EAA/NEAA was 0.59 and 0.60, respectively, and the EAAI was 47.71 and 48.55, respectively. The content of oleic acid, linoleic acid and palmitic acid of Xiehe CoixⅠwas 32.2,20.5 and 6.8 mg/g, respectively, which was higher than that of Xiehe CoixⅡ.3. Yield components and the regulation of endogenous hormonesCorrelation analysis and path analysis of yield components showed that aboveground biomass, panicles per plant, number of tillers per plant were the most relevant components of coix. Principal component analysis showed that the spike number per plant, biomass, number of tills of Xiehe CoixⅠwere high, while the grains per panicle and kernel weight were medium. As to Xiehe CoixⅡ, the spike number per plant, biomass, number of tills and grains per panicle were lower, as compared with I. Thus Xiehe CoixⅠwas more productive.The level of endogenous hormone IAA/ZR of different coix stains was significantly different. Throughout the tiller development process, IAA/ZR ratio of Xiehe CoixⅠwas always higher than CoixⅡ. The difference was most significantly at the beginning and ending period of tiller development. Thus there was a close relationship between the endogenous hormone and the development of tillers.4. Study on photosynthetic and chlorophyll fluorescence characteristics of coixStudy on photosynthetic showed that, at seedling and growth stage, the content of chlorophyll a of Xiehe CoixⅠwas lower than that ofⅡ, but the content of carotenoids was higher thanⅡ. The ratio of a/b of I was decreased by increasing the content of chlorophyll b, thus the light captured by leaves was increased. At maturity stage, the content of chlorophyll a of I was increased significantly.The diurnal change of net photosynthetic rate (Pn) of coix showed a single peak curve. In the morning, the Pn increased significantly with the increase of photosynthetic active radiation, and CoixⅠincreased faster thanⅡ. The Pn reached the maximum at 12:00 (Coix I:18.2μmol CO2 m-2s-1; CoixⅡ:18.9μmol CO2 m-2s-1), then deceased gradually and reached the lowest at sunset. Responses of net photosynthetic rate to light for coix showed that the light compensation point (LCP) of CoixⅠ(24.0μmol m-2s-1) was lower and the light saturation point (LSP) (1197μmol m-2s-1) was higher, thus the using range of light intensity was wider, when compared with CoixⅡ(LCP:50.0μmol m-2s-1, LSP:931μmol m-2s-1).The Fo, Fv/Fm,ΦPSII and qP of CoixⅠwere higher thanⅡ, which means that the PSII reaction center of Coix I’s leaves were more active, with higher electron transport activity and light energy conversion efficiency, as compared with CoixⅠ. However, the NPQ of CoixⅡwas higher thanⅠ, indicated that the potential heat dissipation capacity of PSII was strong, and can effectively prevent the damage on the photosynthetic apparatuscaused by excess light.5. Determination of antioxidant enzyme activitiesAt seedling stage, the activities of SOD and APX of CoixⅡwere significantly higher thanⅠ, but the activities of POD, CAT, GR, anthocyanins and flavonoids were significantly lower. At growth stage, the activities of SOD, POD and APX of CoixⅡwere significantly higher thanⅠ, but the activities of CAT, anthocyanins and flavonoids were significantly lower thanⅠ. At flowering stage, the activities of SOD, POD and APX of CoixⅡwere significantly higher than I, but the activities of GR, anthocyanins and flavonoids were significantly lower thanⅠ. From these results, we concluded that CoixⅠresist free radicals mainly through the antioxidant enzymes, while CoixⅠmainly through anthocyanins and flavonoids.6. Fertizilation, planting density and planting dateThe differences of yield and its components of coix treated with different fertizilations levels and combinations were significant. The yield of CoixⅠtreated with N1P2K2 was the highest (284.4 kg/667m2), the conresponding output value was 2491 yuan/667m2. The equations between yield and dosage of single-fertizilation application were established. The economic optimal dosage of N, P and K applied on CoixⅠwas 6.64, 5.38, and 6.63 kg/667m2. Since the increase of yield of CoixⅠwas mainly through the increase of grain number and seed setting rate, and P and K were the important fertizilation to increase the yield of CoixⅠ, the results of fertizilation uptake analysis indicated that the aborption of P and K of CoixⅠcould be inhanced by reducing the application of N.Effects of different planting densities and planting date on yield and its components in coix were studies using two-factor randomized block design. The results showed that the difference of yield and its components affected by plant densities and planting dates were both significantly. The optimal planting density was 60cm×80cm spacing-row, with respects to increasing economic benefits of coix. While the planting date delayed 20 days, the yield of Xiehe CoixⅠwas 8.2% decreased, and was mainly affected by the number of tillers. Considering the increased consumption of human and irrigation, and the decrease of the content of triolein when planting early, the optimal planting date in Beijing was around mid-May.更多还原