苦瓜(Momordica CharantiaL.)MADS-box基因BAG启动子的克隆分析及表达载体的构建

作者提取苦瓜基因组总DNA,构建了DNA步移文库,并根据已克隆并公布的苦瓜MADS box基因BAG的基因序列设计引物,通过染色体步移技术克隆出BAG基因起始密码子上游调控序列BAGP.对BAGP的鉴定和分析表明其具备大多数高等植物启动子的保守元件,预测它对BAG基因的表达具有一定的作用.为鉴定BAG基因的基本启动子元件,将基因5′侧翼序列做缺失片段分析,利用PCR方法从BAGP中得到3个大小不等两端带有HindⅢ,BamHⅠ酶切位点的片段BAGP1,BAGP2和BAGP3,定向插入载体pMGFP4(pBI221改建,报告基因为GFP)中,取代原有的CaMV35S启动子,构建了由驱动报告基因GFP的植物表达载体BAGPV1,BAGPV2和BAGPV3.

Comparisons between Phenolic Compounds and Antioxidation of Momordica charantia L. in Different Varieties

[Objective] The aim was to compare contents of free and bound polyphenol and antioxidation of balsam pear in different varieties. [Method] Fourteen varieties of balsam pear were chosen to analyze total contents of free or bound phenol and components of free phenol. FRAP （Freeic reducing/antioxidant power）, DPPH （1,1diphenyl-2-picrylhydrazy）, and ABTS ＋· （2, 2’-azinobis-3-ethylbenzothiazoline-6-sulphonic acid diammonium salt） were used to measure antioxidation and analyze relationship between polyphenol content and antioxidation. [Result] Among the fourteen varieties of balsam pear, free, bound and total phenols were 157.58 -382.92, 6.46 -54 and 175.27 -413.79 mg GAE/100 g DW; coefficients of variance were 23.50% , 61.04% and 21.58%, respectively; free phenol accounted for 91.34% of total phenol and bound phenol accounted for 8.66% ; contents of total flavone for the fourteen varieties were from 8.97 to 18.22 mg CE/100 g DW and the coefficient was at 22.80%; vanillic aldehyde acid, epicatechin and rutin differed in contents among different varieties, which changed in 1.83-9.29, 35.17-114.52 and 0.91-4.53 mg/100 g DW and the coefficients were 43.85%, 26.97% and 33.09%; FRAP antioxidation was at 272.16 -713.32 mg TE/100 g DW and coefficient at 27.67% ; IC 50 of DPPH and ABTS ＋· clearance were 11.43-34.14 and 21.57-119.71 mg GAE/100 g DW and coefficients were at 35.10% and 63.75% , accordingly; content of total phenol was of extremely positive correlation with FRAP （P0.01） and of extremely negative correlation with IC 50 of DPPH and ABTS ＋ · clearance （P0.01）. [Conclusion] Contents of phenolic compounds and antioxidation are of significant genotype differences; and phenolic substance and antioxidation are mainly in free form and phenolic substance is the major basis for antioxidation.

Identification and Evaluation of Cold Tolerance in Bitter Gourd (Momordica charantia L.) at Bud and Seedling Stages under Different Temperature Conditions

[ Objectives] This study was conducted to screen the temperature index most suitable for identification of cold tolerance in bitter gourd （Momordica charantia L. ） at bud and seedling stages. [ Methods] With six different bitter gourd germplasms as experimental materials, the cold tolerance at bud and seedling stages were identified and evaluated. [ Results] At 18℃ , the largest change range of germination potential among different gernlplasms was 11.3% -96.0%, and the largest change range of germination rate was 13.3% - 100.0% ; and the six germplasms could be divided into three grades of cold tolerance. Therefore, 18 9C is an ideal temperature for the identification of cold tolerance in bitter gourd germplasms at bud stage. At 20℃, the cold tolerance in bitter gourd germplasms at bud stage could also be divided into three grades, and the change ranges of germination potential and germination rate were larger than 50.0%, so 20℃ could serve as the candidate temperature for the identification of cold tolerance in bitter gourd germplasms at bud stage. After 1 d of treatment at 6℃, the six bitter gourd germplasms could be divided into three grades, and the cold injury index had the largest range of 20.31 - 84.38 and could serve as the temperature index for the identification of cold tolerance in bitter gourd at seedling stage. [ Conclusions] This study will provide reference for the identification of cold tolerance in bitter gourd at bud stage and seedling stage.

Fusarium Wilt Resistance Identification and Genetic Analysis of Momordica charantia L. Germplasm

Fusarium wilt seriously affects the yield and quality of Momordica charantia L.Breeding resistant varieties is the most economical and effective measure to control Fusarium wilt in M.charantia L.This study had comprehensively conducted pathogen identification of Fusarium wilt,resistance evaluation of M.charantia L.germplasm to Fusarium wilt,and genetic analysis of Fusarium wilt resistance in M.charantia L.,so as to provide reference for breeding resistant varieties and accelerating the breeding process.

Callus and Adventitious Buds Formation from Stem Explants of Balsam Pear （Momordica charantia L.）

The effects of sterilization pretreatment, sampling time and growth regulators on callus formation, explant contamination and adventitious bud induction from stems of balsam pear （Momordica charantia L.） were investigated. Sterilized for 6 rain with 0.1% （w/v） mercuric chloride solution is an essential sterilization method for stems. June and July proved to be better for material taking, followed by May. The callus formation rate of balsam pears reached the highest when medium added with IBA 1.0mg/L and BA 2.0 mg/L, while the optimum medium for adventitious bud induction was MS added with IBA 0.5mg/L and BA 4.0 mg/L. And it will provide theoretical and technical basis for rapid propagation and breeding.

Morphological Characteristics of Floral Organ at Different Microspore Developmental Stage in Balsam Pear （Momordica Charantia L.）

The relationship between microspore developmental stages and morphological characteristics of floral organ in balsam pear was studied. The results showed that flower buds developed a series of morphological changes at different microspore developmental stages, and morphological characteristics of floral organ were significantly different when sampling from different part or cultivar of balsam pear. Anther at late-uninucleate stage was best for culture, in which stage flower buds swelled obviously, and white floss on the surface reduced. In addition, calyxes were obvious but did not spread, and the most of anthers were light green.

Effects of Light-Emitting Diode (LED) Red and Blue Light on the Growth and Photosynthetic Characteristics of <i>Momordica charantia</i>L.

With andromonoecious Momordica charantia L. (bitter gourd) as material, three light qualities (50 μmol·m-2·s-1) including white LED light (WL), blue monochromatic light (B, 465 nm), and red monochromatic light (R, 650 nm) were carried out to investigate their effects on seed germination, physiological and biochemical parameters, sex differentiation and photosynthetic characteristics of bitter gourd. The results showed that compared to the WL treatment, the R treatment significantly promoted seed germination, seedling height elongation and soluble sugar content, the B treatment significantly increased seedling stem diameter, reducing sugar content and soluble protein content, the R and B treatments both significantly reduced sucrose content, but their POD activity showed no significant difference. Compared with the R treatment, the B treatment significantly increased the total female flower number and female flower nod ratio in 30 nods of main stems. The study of photosynthetic characteristics found that the R and B treatments could effectively increase the stomatal conductance (GS) of leaves, significantly improved the net photosynthetic rate (Pn) compared to the WL treatment, and the effect of the B treatment was better. Compared to the R and WL treatments, the B treatment increased the maximum photosynthetic rate (Pmax), apparent quantum efficiency (AQE) and light saturation point (LSP), and reduced the dark respiration rate (Rd) and light compensation point (LCP) of the leaves. Fit light response curves showed that the adaptability and utilization of weak light in bitter gourd were middle or below, but it showed higher adaptability and utilization of strong light. Thus, it suggests that Momordica charantia is a typical sun plan with lower Rd. In summary, it is concluded that blue light has a positive effect on the seed germination, seedling growth, sex differentiation and improving the photosynthetic performance, and this will lay the foundation for artificially regulating optimum photosynthesis using specific LEDs wavelength, and help to elucidate the relationship how light quality influences the sex differentiation of plant.

Field Evaluation on Agronomic Characteristics of Newly Introduced Bitter Gourd(Momordica charantia L.)Accessions in Thua Thien Hue Province,Vietnam

The main objective of this study was to evaluate ability of growth and yield of introduced bitter gourd accessions in winter-spring 2016-2017 in Thua Thien Hue province.A total of seven accessions were used in this study.Of these,six accessions were provided by the World Vegetable Center(AVRDC),namely,AVRDC 1329,AVRDC 1330,AVRDC 1331,AVRDC 1333,AVRDC 1334,AVRDC 1335 and one Vietnam local accession(control)from Dien Hai commune,Phong Dien district,Thua Thien Hue province.The results showed that experiment accessions can grow under Thua Thien Hue conditions.AVRDC 1329,AVRDC 1330,and AVRDC 1335 were considered as displaying good growth and development ability.Of those,AVRDC 1330 was the most suitable to consumer as regards to appearance and bitter taste.AVRDC 1330,the control check and AVRDC 1331 had the high actual yield with 16.57,10.65 and 7.88 tons/ha,respectively,and these two introduced accessions can be used for breeding and cultivation under local condition.

A new cucurbitane triterpenoid from Momordica charantia

One new cucurbitane-type triterpenoid saponin, 5β,19-epoxycucurbita-6,23-diene-3β,19,25-triol-3-O-β-D-allopyranoside （1）,named momordicoside P was isolated from the fresh fruits ofMomordica charantia. The structure of the saponin was elucidated by spectral methods, including 2D-NMR spectra.

基于HPLC指纹图谱评价不同来源苦瓜叶的皂苷质量特征

[目的]分析不同来源苦瓜叶皂苷的质量特征,以期为苦瓜叶的质量评价和杂交利用提供依据。[方法]利用高效液相色谱法(HPLC)建立4个来源79份种质的苦瓜叶皂苷指纹图谱,通过相似度评价、变异分析和主成分分析综合评价苦瓜叶的皂苷质量,并进一步通过回归分析和偏最小二乘分析筛选苦瓜叶的质量评价指标和差异标志物。[结果]建立的指纹图谱共标定了16个共有峰,其中UV指纹图谱12个、ELSD指纹图谱4个,与对照图谱的相似度分别在0.848~0.997和0.741~1.000之间,平均变异系数为46.59%,变异幅度在30.24%~71.10%之间;群体间的变异程度以山东群体最高,变异幅度在33.03%~55.10%之间。主成分分析表明,前5个主成分累计贡献率达91.234%,反映了苦瓜叶皂苷指纹图谱的大部分信息。主成分载荷值的综合得分表明,不同来源以及相同来源不同种质苦瓜叶的皂苷质量存在差异,KG 4表现最优(2.199),其次为KT 12(2.179)、KT 28(1.897);4个群体以山东群体表现最优(0.327),其次为福建和广东群体,台湾地区群体表现最差(-0.501)。回归分析筛选出7个色谱峰,可作为评价苦瓜叶质量的鉴定指标,但偏最小二乘分析建立的模型预测能力差,不同来源苦瓜叶皂苷无明显的特征差异。[结论]山东群体苦瓜叶的皂苷质量较其他3个群体表现更好。不同来源苦瓜叶皂苷无明显的地域特征。色谱峰4、5、6、7、11、12、16所代表的成分可作为苦瓜叶皂苷质量评价的鉴定指标。

苦瓜种质资源遗传多样性的AFLP分析

对36份苦瓜种质资源进行AFLP-银染分子标记技术扩增,进行遗传多样性和亲缘关系评价分析。筛选8个多态性高、分辨力强的E+3/M+3引物组合对36份供试材料的基因组DNA扩增,共获得条带数1142条,多态性条带数992条,扩增效率87%,平均每对引物扩增条带数142.75条。其中,以E-ACA/M-CAG引物的扩增效率最高,达到91.79%;其次是E-AAG/M-CTA,达到90.30%;扩增效率最低的引物是E-ACA/M-CTT,为82.31%。表明供试材料在DNA水平上酶切位点的分布存在广泛的变异。各供试材料相似系数介于0.64～0.89;UPGMA分析将36份资源从变种间水平分为2个类群。

苦瓜AFLP和SRAP的PCR反应体系优化及应用比较

【目的】优化苦瓜AFLP和SRAP的PCR反应体系,比较这两种分子标记在苦瓜应用中的优劣,为苦瓜突变体筛选提供参考。【方法】以8份苦瓜种质为材料,对AFLP和SRAP反应体系中各反应因素浓度进行筛选优化;分别选取5对条带清晰、重复性好的AFLP和SRAP引物对8份苦瓜材料进行扩增,比较其多态性。【结果】在AFLP反应体系中,在0～10倍稀释范围内,以连接产物稀释5倍时反应效果较好;将预扩产物稀释30倍进行选择性扩增为宜。在SRAP反应体系中,Mg2+浓度为2.5mmol/L、dNTP浓度为0.2mmol/L时,反应效果较好。多态性分析结果显示,5对AFLP引物共扩增出145条带,多态性条带有18条,多态性比率为12.41%;5对SRAP引物扩增条带数为109条,其中多态性条带数有9条,多态性比率为8.25%。【结论】AFLP和SRAP两种分子标记技术均可以很好反映苦瓜种质资源的遗传多样性,但AFLP扩增产物的平均多态性高于SRAP,能够比较稳定地筛选出差异条带。

苦瓜皂苷对成骨细胞增殖和ALP的影响

研究苦瓜皂苷对体外培养的新生SD乳鼠成骨细胞增殖和ALP的影响。以1×10-8～1×10-4mol/L浓度梯度的苦瓜皂苷的MEM含药培养液对新生SD乳鼠24h内颅盖骨进行体外细胞培养,分别用MTT法和PNPP法检测成骨细胞的增殖和碱性磷酸酶(alkaline phosphatase,ALP)活性。结果表明:苦瓜皂苷具有时间和剂量依赖性地促进成骨细胞增殖和提高成骨细胞ALP活性。

HPLC-ELSD测定苦瓜皂甙的含量

本文用HPLCELSD法测定苦瓜中皂甙B的含量。色谱条件:色谱柱ZorbaxSBC18,4.6×150mm;检测器ELSD(EvaporativeLightScatteringDetector);柱温30℃;流动相:甲醇水(70:30);流速1.0mL/min,N2压力:2.0×105Pa。皂甙B的保留时间在为7.295min,在浓度为0.1058～5.290μg/μL之间有很好的线性关系(R2=0.9918)。平均加标回收率为100.27%(n=3),RSD=2.83%。

苦瓜外观性状AFLP反应体系的建立及优化

为探讨苦瓜性状遗传多样性奠定基础,通过对酶切方法的比较研究,建立并优化了苦瓜AFLP反应体系,采用TaqI/VspI或EcorI/MseI2种限制性内切酶酶切组合对的苦瓜(Momordica charantia.L.)的遗传多样性进行研究,探索适合其AFLP的双酶切体系。结果表明EcorI/MseI内切酶进行双酶切时酶切效果较好并且扩增出的条带带纹清晰,扩增信号强度基本一致符合研究需求,并初步筛选得到了32对清晰、多态性高的AFLP引物。说明最适宜的酶切方法是使用EcorI/MseI组合进行双酶切,通过重复试验证明了该体系稳定可靠,可用于苦瓜性状遗传多样性的分析。

不同发育阶段苦瓜皂苷变化特征及相关性分析

[目的]本文旨在探究苦瓜皂苷坐果期间不同发育阶段的变化特征、差异性以及相关性,为苦瓜的功能性育种、最佳采收期确定及品质调控提供科学依据。[方法]以自主选育的苦瓜‘明绿5号'为研究对象,通过香草醛法、高效液相色谱-质谱法、离子发射光谱仪等测定苦瓜皂苷的组成、含量及其理化因子,并对结果进行化学计量学分析。[结果]不同发育阶段苦瓜总皂苷含量差异极显著(P<0.01),总体呈下降趋势,含量为3.14~10.64 mg·g^(-1),而单果总皂苷含量呈上升趋势,含量为9.03~77.14 mg。特征图谱标定共有峰55个,包含苦瓜皂苷的大部分信息,不同发育阶段共有峰峰面积相对标准偏差(RSD)的变幅为14.68%~193.01%,其中变幅在50%以上的共有峰比例达85.45%。聚类分析与主成分分析结果一致,均将幼果期3个批次聚成一类,膨大期聚为一类,3个成熟期聚为一类。通过偏最小二乘判别分析,苦瓜青熟期至完熟期差异较小,但与幼果期、膨大期差异显著,经质谱验证,最终确定了10个特征差异代谢物。相关性分析表明,在坐果期间,差异代谢物总体与总皂苷、果形指数、氮元素、磷元素和铜元素呈极显著正相关(P<0.01),与果实纵径、果实横径、单果重、果肉厚和含水率呈极显著负相关(P<0.01)。[结论]不同发育阶段苦瓜皂苷组成较稳定,总皂苷及其组分含量有显著性差异。转色期可作为以皂苷收获为目标的最佳采收期。苦瓜皂苷L、苦瓜皂苷K等10个单体皂苷组分是不同发育阶段的主要活性单体,其变化与总皂苷、果形指数以及氮、磷、铜元素密切相关。

苦瓜杂交一代种子纯度InDel分子标记鉴定技术研究

以6份苦瓜自交系及其配组的3个苦瓜杂交一代组合为试验材料,对6份苦瓜自交系进行高深度重测序,鉴定获得覆盖苦瓜全基因组的InDel变异位点信息,并针对InDel变异位点设计引物进行PCR扩增验证,成功开发3个苦瓜杂交一代组合的各自特异多态性InDel分子标记,将多态InDel分子标记应用于苦瓜杂交一代种子纯度鉴定。结果表明,InDel分子标记纯度鉴定结果与田间种植鉴定结果一致,初步建立了苦瓜杂交一代种子纯度InDel分子标记鉴定技术体系。

苦瓜皂苷L对异丙肾上腺素诱导的大鼠H9c2心肌细胞肥大的作用研究

目的探究苦瓜皂苷L对异丙肾上腺素诱导的大鼠H9c2心肌细胞肥大的保护作用及机制。方法 (1)体外培养大鼠H9c2心肌细胞,采用MTT法评价不同浓度的异丙肾上腺素(0.625、1.25、2.5、5、10μmol·L-1)和不同浓度的苦瓜皂苷L(1.625、3.125、6.25、12.5、25.0μg·mL^(-1))对心肌细胞活力的影响。(2)采用异丙肾上腺素诱导心肌细胞肥大模型。实验分为正常组、模型组、苦瓜皂苷L干预组和苦瓜皂苷L单独组。分别通过细胞拍照结合ImageJ软件测量、荧光定量PCR法考察苦瓜皂苷L 12.5μg·mL^(-1)对异丙肾上腺素诱导的心肌细胞表面积和心肌肥大相关胚胎基因ANP、β-MHC及α-SKA表达的影响。在此基础上,选择参与甘油磷脂代谢的酶VI组磷脂酶A2(PLA2G6)、二酰甘油激酶ζ(DGKZ)、甘油磷酸胆碱磷酸二酯酶1(GPCPD1)为指标,评价苦瓜皂苷L对异丙肾上腺素诱导的心肌细胞上述3种酶基因表达的影响;进一步以PLA2G6和DGKZ蛋白为指标,采用Western Blot法考察苦瓜皂苷L对异丙肾上腺素诱导的心肌细胞PLA2G6和DGKZ蛋白表达的影响。结果 (1)与正常组比较,异丙肾上腺素(0.625~10μmol·L-1)和苦瓜皂苷L(1.625~25μg·mL^(-1))对心肌细胞活力没有明显影响(P>0.05)。本研究选用异丙肾上腺素10μmol·L^(-1)和苦瓜皂苷L 12.5μg·mL^(-1)进行实验。(2)与正常组比较,模型组心肌细胞表面积增加(P<0.05),心肌肥大相关胚胎基因ANP、β-MHC、α-SKA表达上调(P<0.05),PLA2G6、DGKZ、GPCPD1 mRNA表达上调(P<0.05),PLA2G6和DGKZ蛋白表达增加(P<0.05);与模型组比较,苦瓜皂苷L干预组能够降低心肌细胞表面积(P<0.05),下调心肌肥大相关胚胎基因ANP、β-MHC、α-SKA表达(P<0.05),下调PLA2G6、DGKZ、GPCPD1 mRNA表达(P<0.05),降低PLA2G6和DGKZ蛋白表达(P<0.05)。结论苦瓜皂苷L可缓解异丙肾上腺素诱导的心肌细胞肥大,可能与其抑制甘油磷脂代谢酶PLA2G6和DGKZ的表达有关。

苦瓜皂苷对人恶性黑素瘤SKMEL-2细胞增殖凋亡及JAK/STAT5通路的影响

目的:探讨苦瓜皂苷对人恶性黑素瘤SKMEL-2细胞生长及JAK/STAT5通路的影响。方法:采用1、2.5、5、7.5、10mg/mL浓度的苦瓜皂苷处理体外培养的人恶性黑素瘤SKMEL-2细胞,以CCK-8法分别在24、48、72h后检测细胞增殖情况,筛选合适药物作用浓度和时间。体外培养的SKMEL-2细胞,随机分为三组:对照组(control)、苦瓜皂苷组及顺铂组,药物处理后,CCK-8检测细胞增殖情况,测定细胞存活率;流式细胞技术检测细胞凋亡情况,测定细胞凋亡率;采用免疫印记(WB)法检测caspase3、Bcl-2、Bax、JAK、p-JAK、STAT5、p-STAT5的蛋白表达水平。结果:药物作用48、72h其抑制作用比24h高,而药物作用48、72h其抑制作用基本相同;浓度为1~5、7.5、10mg/mL的苦瓜皂苷都能抑制SKMEL-2增殖,其抑制作用随浓度升高而增强,但在药物作用48、72h后,浓度达到5mg/mL时抑制作用趋于稳定。选择5mg/mL浓度的苦瓜皂苷处理SKMEL-2细胞48h用于后续实验;药物处理后,与control组比较,苦瓜皂苷组及顺铂组细胞相对存活率、p-JAK、p-STAT5、Bcl-2表达明显降低(P<0.05),细胞凋亡率、caspase3、Bax表达明显升高(P<0.05),JAK、STAT5表达无明显变化,差异无统计学意义(P>0.05);苦瓜皂苷组与顺铂组相比,各指标无明显变化,差异无统计学意义(P>0.05)。结论:苦瓜皂苷对人恶性黑素瘤SKMEL-2细胞的生长有抑制作用、对其凋亡有促进作用,下调JAK/STAT5通路活性,上调凋亡蛋白caspase3、Bax表达,抑制抗凋亡蛋白Bcl-2表达可能是其作用机制。

Long-Term Treatment with an Herbal Formula MCC Ameliorates Obesity-Associated Metabolic Dysfunction in High Fat Diet-Induced Obese Mice: A Comparative Study among MCC and Various Combinations of Its Constituents

Obesity has been found to be associated with increased incidence of various metabolic disorders. Anti-obesity interventions are therefore urgently needed. An earlier study has demonstrated that treatment with an herbal formula MCC, which comprises the fruit of Momordica charantia (MC), the pericarpium of Citri reticulate (CR) and L-carnitine (CA), reduced the weight gain in high fat diet (HFD)-fed mice. In the present study, we investigated the effect of long-term treatment with MCC (6 g/kg/day × 40 doses) and various combinations of its constituents in HFD-fed female ICR mice. Body weight change was monitored during the course of the experiment. Total and differential adiposity, plasma lipid contents, metabolic enzyme activities and mitochondrial coupling efficiency in skeletal muscle were measured. Glucose homeostasis was also assessed. Results showed that HFD increased the body weight, total and differential adiposity, and plasma lipid contents as well as impaired metabolic status in skeletal muscle and glucose homeostasis. MCC and all combinations of its constituents reduced the weight gain in HFD-fed mice, which was accompanied with an improvement on glucose homeostasis. While MC, CA and CR independently suppressed the HFD-induced weight gain in mice, MC seems to be the most effective in weight reduction, all of which correlated with the induction of mitochondrial uncoupling in skeletal muscle. Only CA and CR, but not MC, significantly reduced the total adiposity and visceral adiposity as well as plasma cholesterol level. However, the two component combinations, MC + CR and MC + CA, decreased the degree of visceral adiposity and plasma cholesterol level, respectively. MCC treatment at 1.5 g/kg (but not a higher dose of 6 g/kg) suppressed visceral adiposity and induced mitochondrial uncoupling in skeletal muscle in HFD-fed mice. The finding suggests that MCC may offer a promising prospect for ameliorating the diet-induced obesity and metabolic disorders in humans.