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.

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.

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.

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.

苦瓜种质资源耐热性评价及其鉴定指标的筛选

本研究以27个苦瓜自交系4叶1心的幼苗为材料,预培养1 d后,置于39℃高温培养,观察其高温胁迫72 h后的热害情况,通过计算热害指数(TDG)筛选评价出高、中、低耐热性的苦瓜种质;从中选取6个不同耐热性的自交系,测定39℃胁迫0 h和48 h的株高(PH)、茎粗(SD)、鲜物质量(SFW)和干物质量(SDW)等形态学指标以及叶片相对叶绿素含量(SPAD)、电导率(REL)、过氧化物酶活性(POD)、过氧化氢酶活性(CAT)、超氧化物歧化酶活性(SOD)、丙二醛含量(MDA)、脯氨酸含量(Pro)和可溶性糖含量(SS)等生理指标;主成分分析上述12个指标的耐热系数(α),综合评价6个自交系材料的耐热性;利用逐步回归分析法建立耐热性评价数学模型获得苦瓜耐热性鉴定指标体系。结果表明:以TDG为依据,27个苦瓜自交系中有耐热型3个,中等耐热型18个,热敏型6个;6个不同耐热性材料的综合评价结果与热害指数鉴定结果基本一致;耐热性的评价数学模型为:Y=-0.426+0.049X1-0.011X2-0.009X3-0.018X4,利用建立的最优回归方程预测供试材料的耐热性,预测值与Y值基本一致。说明SFW(X1)、POD活性(X2)、CAT活性(X3)和SS含量(X4)这4个指标的耐热系数可以快速准确地评价苦瓜耐热性差异,可用作苦瓜耐热性鉴定指标。本研究筛选出苦瓜耐热种质并构建苦瓜耐热性评价体系,为海南夏季耐热苦瓜新品种选育提供依据。

苦瓜枯萎病生防细菌ZB36的分离鉴定及其定殖特性研究

【目的】筛选并鉴定对苦瓜枯萎病具有稳定拮抗活性的生防菌株,明确其在苦瓜植株上的定殖特性。【方法】从山东省淄博市耕作地采集土壤样品,以苦瓜枯萎病菌为靶标,通过梯度稀释涂布平板法分离、纯化,获得1株具有稳定拮抗活性的纯培养细菌,命名为ZB36。根据菌株形态学特征、生理生化特性,以及16S rRNA基因和gyrA保守基因序列分析进行菌株鉴定。通过抗生素抗性标记法获得遗传特性和拮抗活性稳定的利福平标记菌株ZB36^(R),并对其在苦瓜上的定殖特性进行探究。【结果】菌株鉴定结果表明,ZB36为贝莱斯芽孢杆菌(Ba-cillus velezensis)。盆栽试验结果表明,ZB36不仅对苦瓜枯萎病具有显著的防治效果,防效高达74.76%,而且对苦瓜植株也具有显著促生作用,接种ZB36后苦瓜地上部鲜重和株高分别提高了28.65%和22.48%。定殖试验结果表明,ZB36在苦瓜根、茎和叶中均能定殖,且在接种后28 d仍能在这些组织中稳定定殖。【结论】贝莱斯芽孢杆菌ZB36不仅对苦瓜枯萎病具有良好的防治和促生效果,同时也具有较强的定殖能力,具备作为苦瓜枯萎病生防细菌资源开发利用的潜力。

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。

苦瓜品质性状主基因+多基因混合遗传效应分析

开展苦瓜(Momordica charantia L.)干物质含量、可溶性蛋白质含量和硝酸盐含量的品质性状研究,分析其遗传机制。以苦瓜雌性系Z-1-4为母本,优质苦瓜高世代自交系2538、5325和5321为父本,构建3个组合的4个世代[P_(1)(亲本)、P_(2)、F_(1)、F_(2)]遗传群体,并结合主基因+多基因混合遗传模型对苦瓜干物质含量、可溶性蛋白质含量和硝酸盐含量进行遗传及相关分析。结果表明,苦瓜干物质含量最适遗传模型为等加性主基因加性+多基因模型,且遗传率较高;苦瓜可溶性蛋白质含量最适遗传模型为负向完全显性主基因+加性-显性多基因模型,遗传率较低,适合在早期世代进行遗传选择。商品瓜硝酸盐含量最适遗传模型为2对加性-显性主基因+加性-显性多基因模型,遗传率也较低。可溶性蛋白质与硝酸盐的遗传率均相对不高,适合在高世代进行表型选择。

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。

热风辅助微波干燥对苦瓜干燥品质和效率的影响

为研究热风辅助微波干燥因素对苦瓜干燥品质和效率影响,以苦瓜红度值a*、水分活度、叶绿素含量和干燥速率作为干燥品质和效率评价指标,通过单因素试验,分析微波功率、热风温度和热风速度等干燥因素对苦瓜品质和干燥效率影响规律,确定干燥因素范围。在此基础上,通过二次正交旋转组合试验,建立预测苦瓜品质和干燥效率二次多项式回归模型。结果表明,四项指标R2分别为0.9055、0.9170、0.8939、0.9805,均大于0.8900,表明回归模型拟合性较高,可较好预测苦瓜干燥品质和效率。利用响应面法确定影响苦瓜热风辅助微波干燥最佳干燥因素为:微波功率900W,热风温度60℃,热风速度20m·s^(-1)。研究可提高苦瓜干燥品质和干燥效率,为促进苦瓜干燥商业化发展提供一定理论指导及技术支持。

苦瓜中葫芦烷型三萜成分及其抗肝纤维化活性的研究

目的研究苦瓜酸水解产物的化学成分及其抗肝纤维化活性。方法用正、反相柱层析等色谱手段进行分离纯化,用波谱技术与文献数据对照鉴定其结构;并用四甲基偶氮唑盐(methylthiazolyl tetrazolium,MTT)法测定部分化合物体外对大鼠肝星状细胞(t-HSC/Cl-6)增殖的抑制作用。结果共分离出10个葫芦烷型三萜类化合物,分别鉴定为:5β,19-epoxy-3β-hydroxy-19(R)-methoxy-27-norcucurbita-6,23(E)-dien-25-one(1);5β,19-epoxy-3β-hydroxy-19(S)-methoxy-27-norcucurbita-6,23(E)-dien-25-one(2);5β,19-epoxy-25-methoxycucurbita-6,23(E)-dien-3β-ol(3);karavilagenin A(4);(23E)-5β,19-epoxycucurbita-6,23,25-triene-3β-ol(5);23E-5β,19-epoxy-cucurbita-6,23-dien-3β,25-diol(6);25-O-methylkaravilagein D(7);(23E)-3β-hydroxy-7β,25-dimethoxycucurbita-5,23-dien-19-al(8);karavilagenin B(9);(19S,23E)-5β,19-epoxy-19-methoxycucurbita-6,23-diene-3β,25-diol(10)。体外活性筛选发现化合物1和化合物2对t-HSC/Cl-6有一定的抑制细胞增殖作用,IC50分别为(25.92±2.63)、(26.22±2.77)μmol·L^(-1)。结论化合物1和化合物2为首次从该植物中分离得到,其对肝纤维化细胞增殖有一定的抑制作用。

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。

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.