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1.
In order to develop an efficient method about protein extraction which is suitable for apple proteomic analysis, four protocols of total protein extraction from apple leaves, which are trichloroacetic acid/acetone precipitation method （TCA）, phenol extraction methanol/ammonium acetate precipitation method, Tris-HCl extraction method and modified Tris-HCl extraction method were compared. The results showed that the modified Tris-HCl extraction method was the most suitable method in protein extraction for two-dimensional electrophoresis （2-DE） from apple leaves based on the highest resolution and more informative spots of 2-DE gels with no apparent vertical or horizontal streaking. 相似文献
2.
Muhammad ARF Sultan LIU Hui CHENG Yu-Feng ZHANG Pei-pei ZHAO Hui-xian 《中国农业科学(英文版)》2013,(7):1123-1129
Proteomic assessment of low-abundance leaf proteins is hindered by the large quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) present within plant leaf tissues. In the present study, total proteins were extracted from wheat (Triticum aestivum L.) leaves by a conventional trichloroacetic acid (TCA)/acetone method and a protocol first developed in this work. Phytate/Ca2+ fractionation and TCA/acetone precipitation were combined to design an improved TCA/acetone method. The extracted proteins were analysed by two-dimensional gel electrophoresis (2-DE). The resulting 2-DE images were compared to reveal major differences. The results showed that large quantities of Rubisco were deleted from wheat leaf proteins prepared by the improved method. As many as (758±4) protein spots were detected from 2-DE images of protein extracts obtained by the improved method, 130 more than those detected by the TCA/acetone method. Further analysis indicated that more protein spots could be detected at regions of pI 4.00-4.99 and 6.50-7.00 in the improved method-based 2-DE images. Our findings indicated that the improved method is an efficient protein preparation protocol for separating low-abundance proteins in wheat leaf tissues by 2-DE analysis. The proposed protocol is simple, fast, inexpensive and also applicable to protein preparations of other plants. 相似文献
3.
A bacterial artificial chromosome （BAC） library is an invaluable resource tool to initiate tea plant genomics research, and the preparation of high molecular weight （HMW） genomic DNA is a crucial first step for constructing a BAC Library. In order to construct a BAC library for enhancing tea plant genomics research, a new method for the preparation of tea pant high molecular weight （HMW） genomic DNA must be developed due to young tea plant leaves and shoots are notably rich in both tea polyphenols and tea polysaccharides. In this paper, a modified method for preparing high quality tea plant HMW genomi~ DNA was optimized, and the quality of tea plant genomic DNA was evaluated. The results were as follows： Critical indicators of HMW DNA preparation were the appearance of the smooth nuclei in solution （as opposed to sticky-gummy） before agarose plug solidification, non-dark colored nuclei plugs after lysis with an SDS/proteinase K solution, and the quality and quantity of HMW DNA fragments after restriction enzyme digestion. Importantly, 1% dissolved PVP-40 and 1% un-dissolved PVP-40 during the nuclei extraction steps, in conjunction with the removal of PVP-40 from the plug washing and nuclei lysis steps, were critical for achieving HWM tea plant DNA suitable for BAC library construction. Additionally, a third PFGE fraction selection step to eliminate contaminating small DNA fragments. The modifications provided parameters that may have prevented deleterious interactions from tea polyphenols and tea polysaccharides. The HMW genomic DNA produced by this new modified method has been used to successfully construct a large-insert tea plant BAC library, and thus may be suitable for BAC library construction from other plant species that contain similarly interfering compounds. 相似文献
4.
5.
YANG Xiu-shi ;WANG Li-jun ;DONG Chuan ;Edmund Man King Lui ;REN Gui-xing 《中国农业科学(英文版)》2014,(12):2807-2815
Ultrasonic-assisted extraction （UAE） of American ginseng polysaccharides （AGP） was investigated using response surface methodology. Three-factor-three-level Box-Behnken design was employed to optimize the ultrasonic power, extraction time and ratio of water to raw material to obtain a high AGP yield. The analysis of variance and response surface plots indicated that ultrasonic power was the most important factor affecting the extraction yield. The optimal conditions were ultrasonic power 400 W, extraction time 71 min, and ratio of water to raw material 33 mL g-1. Under these conditions, the yield of AGP was 8.09%, which was agreed closely to the predicted value. Gas chromatography （GC） analysis showed that AGP was composed of arabinose, rhamnose, galactose, glucose, and galacturonic acid. Fourier transform infrared spectra revealed the general characteristic absorption peaks of AGP. In addition, AGP exhibited good immunostimulating activities by up-regulating the production of nitric oxide and cytokines. Compared with hot water extraction, UAE required shorter extraction time and gave a higher extraction yield, without changing the structure and immunostimulating activity of AGP. The results indicated that UAE could be an effective and advisable technique for the large scale production of plant polysaccharides. 相似文献
6.
ZHANG Yong-qiang DING Wei ZHAO Zhi-mo WU Jing FAN Yu-hu 《中国农业科学(英文版)》2008,7(5):577-584
The aim of this study was to determine the best extraction technique, the most suitable solvent, the optimal plant parts, and the acaricidal activities of Artemisia annua L. The petroleum ether （30-60℃）, petroleum ether （60-90℃）, ethanol, acetone, and water parallel and sequenced extracts were obtained from the leaves, stems and roots of different period of A. annua L. in April, May, June, July and September respectively. And then the acaricidal bioactivities against Tetranychus cinnabarinus of all extracts were determined by the slide-capillary method in the laboratory. The results indicated that the acaricidal bioactivities elevated as the development of A. annua plant at the concentration of 5 mg mL-L The general tendency exhibited the sequence of July 〉 June 〉 May 〉 April, but September decreased comparing to July. However, the most effective extracts in five months were all acetone parallel extract of A. annua leaf, and the corrected mortalities treated after 48 h ranged from 74 to 100%. The median lethal concentrations （LC50） against T. cinnabarinus of acetone parallel extracts ofA. annua leaves in September, July, June, May and April were 0.5986, 0.4341, 0.8376, 0.9443 and 1.3817 mg mL^-1, respectively, treated after 48 h. The 13 groups were isolated from acetone extracts ofA. annua leaves in July by column chromatography, both the 1 lth and 12th groups exhibited strong bioactivities. The median lethal concentrations of the 1 lth and 12th groups against T. cinnabarinus were 0.3683 and 0.1586 mg mL^-1, respectively. The acetone parallel extract ofA. annua leaf in July was the most toxic to T. cinnabarinus and the corrected mortality was 100% after 48 h. The acetone parallel extract of the 1 lth and 12th groupswere the most active components, acted as the emphases in further study. 相似文献
7.
The extraction of leaf protein from
modern-slam was studied with the thermal method, acid-base method, acidified thermal
method and fermented acid one. The experiment results showed that the acidified thermal
one (pH=4.0，72℃) is available to get the protein content of the product 63.23%, the
ratio of extraction 36.09% and the ratio of obtaining 1.82%. 相似文献
8.
[Objective] The experiment aimed to study an efficient method of Nuclei extraction of cotton and provided technical support for constructing large-insert genomic library and sequencing complete genome. [Method] The cotton cotyledons germinated in dark moisture chamber for one week were chopped with a sharp sterile scalpel in a Petri dish which contained ice-cold nucleus isolation buffer (10 mmol/L MgSO4, 5 mmol/L KCl, 0.5 mmol/L HEPES, 1 mg/ml DTT, 0.25% Triton X-100 and 2% PVP40), then the nuclei were collected after selected through 100, 50 and 30 μm nylon meshes and centrifugation. Meanwhile, the tender leaves and cotyledons with different germination time in dark were treated by grinding method and sharp scalpel method. [Result] The chopping with a sharp scalpel method was very simple and rapid, which did not need grind and mercaptoethanol treatment and the successful extraction rate was 100%.[Conclusion] An efficient method of nuclei extraction of cotton with simple, high efficiency, rapid reaction and poison free were established. 相似文献
9.
大白菜基因组DNA的提取及AFLP反应体系的建立(英文) 总被引：1，自引：0，他引：1
[Objective] The obtained clear AFLP fingerprint of Chinese cabbage provided basis for studies on the molecular markers of Chinese cabbage cultivars and the phylogenetic relationship among Chinese cabbage cultivars. [Method] With the test materials of leaves of Chinese cabbages, the high-quality total DNA from leaves of Chinese cabbages was extracted by the modified CTAB method. DNA restriction-ligase reaction, pre-amplification and selective amplification were optimized, and the AFLP silver-staining reaction system for Chinese cabbage was established. [Result] The quality of DNA template influenced restriction enzyme digestion and the subsequent ligase amplification reaction, while the modified CTAB extraction method could be used in AFLP analysis of Chinese cabbage to obtain a clear AFLP fingerprint. The optimum conditions for restriction enzyme digestion of genomic DNA from Chinese cabbage were as follows: 150 g DNA template, 12.5 μl reaction volume, 1.25 U Eco R Ⅰ, 1.25 U Mse Ⅰ and 5×Reaction Buffer with 4 h at 37 ℃. The ligation reaction with 2.5 h at 20 ℃ was the optimum condition. Six pairs of primers including E-AAC/M-CAG, E-AAG/M-CAC, E-ACA/M-CTG, E-ACT/M-CAC, E-ACT/M-CTT and E-ACT/M-CTC all had its own stable and clear patterns. [Conclusion] With abundant bands and high polymorphism, AFLP selective amplification is an efficient molecular marker for genomic polymorphism of Chinese cabbage. 相似文献
10.
Artificial Neural Network to Predict Leaf Population Chlorophyll Content from Cotton Plant Images 总被引：2，自引：0，他引：2
SUO Xing-mei JIANG Ying-tao YANG Mei LI Shao-kun WANG Ke-ru WANG Chong-tao 《中国农业科学(英文版)》2010,9(1):38-45
Leaf population chlorophyll content in a population of crops, if obtained in a timely manner, served as a key indicator for growth management and diseases diagnosis. In this paper, a three-layer multilayer perceptron （MLP） artificial neural network （ANN） based prediction system was presented for predicting the leaf population chlorophyll content from the cotton plant images. As the training of this prediction system relied heavily on how well those leaf green pixels were separated from background noises in cotton plant images, a global thresholding algorithm and an omnidirectional scan noise filtering coupled with the hue histogram statistic method were designed for leaf green pixel extraction. With the obtained leaf green pixels, the system training was carried out by applying a back propagation algorithm. The proposed system was tested to predict the chlorophyll content from the cotton plant images. The results using the proposed system were in sound agreement with those obtained by the destructive method. The average prediction relative error for the chlorophyll density （μg cm^-2） in the 17 testing images was 8.41%. 相似文献
11.
Xiaoliang Xiang Shuju Ning Xia Jiang Xiaogui Gong Renlei Zhu Lanfang Zhu Daozhi Wei 《Frontiers of Agriculture in China》2010,4(4):416-421
An efficient protein extraction method for two-dimensional gel electrophoresis (2-DE) from plant samples is usually challenging
due to the low protein content and high level of interfering compounds. Proteomic analyses of rice (Oryza sativa L.) roots are limited by the lack of an efficient protein extraction method. To establish an effective protocol of protein
extraction suitable for 2-DE analysis in rice roots, we evaluated three protein extraction methods (trichloroacetic acid [TCA]/acetone,
Mg/NP-40/TCA, and tris-base/acetone). Our results showed that the Mg/NP-40/TCA extraction method had the highest protein yield
and is the best resolution of protein separation among the three methods. The TCA/acetone method exhibited clear protein profiles
and detected more protein spots with the highest intensity in the region of high Mr (above 45 kDa) than the other methods.
However, this method was unable to detect proteins with low-Mr (less than 24.0 kDa). The Tris-base/acetone method showed the
poorest resolution of protein separation. Our results suggest that the Mg/NP-40/TCA method was the most effective among the
three methods and may provide enhanced proteomic information for rice and other crop roots. 相似文献
12.
摘要：C-乳清是胶乳的主要组分,就双向凝胶电泳技术而言,目前仍无有效的蛋白提取方法,这严重制约了双向凝胶电泳技术在橡胶树蛋白组分析上的应用。本文选取3种方法提取C-乳清蛋白,通过对蛋白产量和提取蛋白的双向凝胶电泳图谱分析系统评价3种方法。就蛋白产量而言,三氯乙酸/丙酮沉淀法产量最高（0.86 mg/ml C-乳清）,三氯乙酸沉淀法次之（0.72 mg/ml C-乳清）,酚提取法最低（0. 66 mg/ml C-乳清）。双向凝胶电泳图谱分析表明：酚提取法可检测到447个蛋白点,图谱背景较暗且横纵向纹理多;三氯乙酸沉淀法可检测到821个蛋白点,图谱背景居中;三氯乙酸/丙酮沉淀法可检测到1052个蛋白点,图谱背景清晰且基本无纹理。综上所述,三氯乙酸/丙酮沉淀法最适合C-乳清蛋白提取。本文首次系统评价C-乳清蛋白提取方法,必将推动橡胶树C-乳清蛋白研究发展。 相似文献
13.
金心黄杨(Euonymus japonica L. cv. aureo-pictus)叶片总蛋白质提取及双向电泳技术 总被引：1，自引：0，他引：1
通过比较2种不同的植物叶片总蛋白质提取方法(三氯乙酸/丙酮法和酚法)和优化双向电泳各试验环节,建立金心黄杨叶片总蛋白质的提取方法,以及可以对其蛋白质组进行质谱分析的双向电泳条件。结果采用优化后的酚法进行金心黄杨叶片总蛋白质的提取,IEF蛋白上样量150μg,SDS-PAGE采用12.5%T凝胶,电泳结束后用考马斯亮蓝G-250染色,Melanie 7.0软件分析后得到约274个可分辨蛋白点。 相似文献
14.
通过比较2种不同的植物叶片总蛋白质提取方法（三氯乙酸／丙酮法和酚法）和优化双向电泳各试验环节,建立金心黄杨叶片总蛋白质的提取方法,以及可以对其蛋白质组进行质谱分析的双向电泳条件。结果采用优化后的酚法进行金心黄杨叶片总蛋白质的提取,IEF蛋白上样量150μg,SDS-PAGE采用12．5％T凝胶,电泳结束后用考马斯亮蓝G-250染色,Melanie 7．0软件分析后得到约274个可分辨蛋白点。 相似文献
15.
采用改良的TCA/丙酮法提取叶片总蛋白,建立了一套适用于甜玉米叶片的蛋白质组学研究方法,并对蛋白溶解液、蛋白上样量、第一向IEF等电聚焦时间等条件进行了优化.用改进的蛋白溶解液提取叶片总蛋白,明显减轻了离子干扰造成的蛋白点横拖;500μg蛋白上样量的检出蛋白点最丰富,可达750个以上;延长8 000 V等电聚焦至30 000 Vh可获得良好的蛋白点分离效果;采用优化条件后的双向电泳参数,结果重复性较高,检出蛋白点的线性回归相关系数可达0.93以上. 相似文献
16.
Proteomic assessment of low-abundance leaf proteins is hindered by the large quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) present within plant leaf tissues. In the present study, total proteins were extracted from wheat (Triticum aestivum L.) leaves by a conventional trichloroacetic acid (TCA)/acetone method and a protocol first developed in this work. Phytate/Ca2+ fractionation and TCA/acetone precipitation were combined to design an improved TCA/acetone method. The extracted proteins were analysed by two-dimensional gel electrophoresis (2-DE). The resulting 2-DE images were compared to reveal major differences. The results showed that large quantities of Rubisco were deleted from wheat leaf proteins prepared by the improved method. As many as (758±4) protein spots were detected from 2-DE images of protein extracts obtained by the improved method, 130 more than those detected by the TCA/acetone method. Further analysis indicated that more protein spots could be detected at regions of pI 4.00–4.99 and 6.50–7.00 in the improved method-based 2-DE images. Our findings indicated that the improved method is an efficient protein preparation protocol for separating low-abundance proteins in wheat leaf tissues by 2-DE analysis. The proposed protocol is simple, fast, inexpensive and also applicable to protein preparations of other plants. 相似文献
17.
以2年生花吊丝竹的扦插苗为研究材料,对2种方法(TCA/酚提结合法和PEG法)提取的蛋白质的2-DE差异进行研究.结果表明:PEG法的5个组分的条带差异明显,全蛋白得到了有效分离,在F3中富集到了2条高丰度蛋白带;2种方法的2-DE图谱的均一性分析发现,PEG法中F3富集了高丰度蛋白,提高了2-DE中蛋白质的检测率和分辨率,PEG法中F1-F5共检测的蛋白总数超过了1700个,但是相邻组存在一定的重叠率,而TCA丙酮/酚提结合法只检测到约571个蛋白点,5个组与NF平均有439个蛋白点匹配,达到77.9%以上;质谱鉴定了5种差异蛋白,生物信息学分析发现这5种蛋白是糖酵解、糖异生和ATP合成的重要辅酶.2种方法的比较发现,PEG法能够将花吊丝竹叶片全蛋白中高丰度蛋白单一富集在16%的组分中,从而显著提高了2-DE的分辨率,再通过质谱技术能够检测到更多的影响生物体生长发育的低丰度蛋白,所以PEG法是一种切实可行的且较为理想的蛋白质提取方法. 相似文献
18.
为了选择适用于水稻根部蛋白质组分析的样品制备方法,以水稻苗期幼嫩根尖为材料,采用TCA/丙酮沉淀法、Tris法、尿素/硫脲法对水稻根部总蛋白质进行提取并比对,通过Bradford法、SDS PAGE电泳技术对蛋白质含量和种类进行分析。结果显示,Tris法提取出的水稻根部蛋白质含量和种类明显少于其他2种方法;尿素/硫脲法和TCA/丙酮沉淀法提取的蛋白质种类和含量都较多,其中,尿素/硫脲法的蛋白质图谱中蛋白条带多,染色深而且很清晰,尤其适用于较低分子量蛋白质的提取,TCA/丙酮沉淀法则适于高分子量蛋白质的提取。因此,进行水稻根部总蛋白质组分析时,可选用尿素/硫脲法。 相似文献
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20.
以‘西域1号,甜瓜苗期根系为材料,分别采用改进的TCA/丙酮沉淀法、Tris/酚提取-甲醇/醋酸铵沉淀法及SDS/酚提取法提取总蛋白,进行SDS电泳和双向电泳分离,扫描后的胶图利用Image Master 2D Elite 6.01软件进行分析,重点分析胶图总点数、匹配点数和差异蛋白点数.结果表明:Tris/酚提取-甲醇/醋酸铵沉淀法得到的总蛋白含量最高;TCA/丙酮沉淀法分离得到的蛋白条带和蛋白点数最少,且对分子量较高的偏碱性区域的蛋白提取效果差,与其他2种方法相比明显丢失了较多的蛋白.因此,进行甜瓜幼苗根系总蛋白质组分析时,可采用后2种方法,其中Tris/酚抽提法更简单易行. 相似文献