亚洲免费av电影一区二区三区,日韩爱爱视频,51精品视频一区二区三区,91视频爱爱,日韩欧美在线播放视频,中文字幕少妇AV,亚洲电影中文字幕,久久久久亚洲av成人网址,久久综合视频网站,国产在线不卡免费播放

        ?

        A New Rapid and Batch-oriented Crushing Method for DNA Extraction from Maize Leaves

        2015-01-18 02:49:44PengfeiLIUQuCHENMuhengZENGXiaomingWANGFengJIANG
        Agricultural Science & Technology 2015年5期
        關(guān)鍵詞:農(nóng)學淺析棉花

        Pengfei LIU,Qu CHEN,Muheng ZENG,Xiaoming WANG,Feng JIANG

        Crop Research Institute,Zhongkai University of Agriculture and Engineering,Guangzhou 510225,China

        Responsible editor:Tingting XU Responsible proofreader:Xiaoyan WU

        Molecular biology has been developed rapidly.Molecular markers (RFLP,SSR,RAPD),genetic engineering technology,cell engineering technology,DNAsequencing technology,DNA chip technology and enzyme engineering technology have been widely applied in plant genetic diversity analysis,genetic map and physical map construction,germplasm evaluation,plant DNA fingerprinting,genetic trait marking and assisted selection[1-8].For the researches of molecular biology,the highly efficient extraction of DNA is the premise and basic link.But for DNA extraction from plants,the grinding of plant leaves is the first step,and is also the most time and effort-consuming step.

        There are currently two main grinding methods in laboratory,con ventional grinding method[8-9]and electric drill grinding method[10].For the conventional grinding method,the sample is mixed with quartz sand in a mortar,and then ground by adding liq uid nitrogen or β-mercaptoethanol.For the electric drill grinding method,the sample is mixed with DNA extraction buffer in a centrifuge tube,and then ground by a high-speed running electric drill.The running electric drill has disadvantages of damaging tubes,wasting samples and consuming a large amount of efforts.Both of the two grinding methods are characterized by time and effort consuming and non-u niformty,which are particularly prominent in DNA extraction from a large number of samples in molecular marker-assisted breeding.

        The inbred lines Zheng 58,PH6WC,Yu 87-1 and Zi 330 were selected for constructing a four-way cross population (Zheng 58/Yu 87-1//PH6WC/Zi330).Based on SSR molecular marker analysis,the genetic linkage map of maize was constructed[10].A new rapid and batch-oriented crushing method was developed for DNA extraction from plant leaves.Moreover,the practicability of extracted DNA in SSR molecular marker analysis was verified.

        Materials and Methods

        Test materials and design

        In autumn of 2010,two cross combinations,Zheng 58/Yu87-1 and PH6WC/Zi 330 were developed in the teaching and research base of Zhongkai University of Agriculture and Engineering in Guangzhou.The F1generations of the two single-cross combinations were cultivated in spring of 2011.The full-sib mating was carried out.The four-way cross population (Zheng 58/Yu 87-1//PH6WC/Zi 330) was constructed.The F2generation (F2:3family)of the four-way cross population was planted in the teaching and research base in Panyuzhongcun Village in spring of 2012.During the seedling stage,the single plant DNA of the F2population was extracted for genotyping and constructing genetic linkage map of molecular marker.

        Crushing of leaves

        According to requirements by test,certain amount (50-100 mg) of fresh plant leaves was weighted and placed in a 2 ml round-bottomed centrifuge tube.A clean steel ball weighing more than 0.5 g was placed in the centrifuge tube.Then the lid of centrifuge tube was covered.All the prepared centrifuge tubes were placed in an antifreeze container and flooded by liquid nitrogen.The antifreeze container was shocked 1-5 min 10-90 s after covering the lid of the container.After then,the lids of the centrifuge tubes were opened,and the steel balls in the centrifuge tubes were sucked out using a magnet.Thus the powder samples were prepared,and they were preserved at 4 ℃.

        DNA extraction

        The DNA extraction was performed using the kit.The plant DNA kit was provided by the Beijing Dingguo Changsheng Biotechnology Co.,Ltd.

        SSR molecular marker detection

        The SSR primer,of which the sequence was uniformly distributed in maize genome,was screened out from the Maize GDB (www.maizgdb.org).The primers,Taq polymerase,dNTP,ddWater,TE buffer were all purchased from the Beijing Dingguo Changsheng Biotechnology Co.,Ltd.

        The PCR reaction system was composed of 1 μl DNA Template (10-20 ng/μl),1 μl Forward Primer (5 μmol/μl),1 μl Reverse Primer (5 μmol/μl),3 μl 10× Taq buffer (containing 20 mM Mg2+),0.6 μl Taqase(2 U/μl),0.6 μl dNTPs (10 mM/μl) and 2.8 μl ddH2O.

        The PCR reaction program was as follows:hot lid temperature of 96 ℃,95 ℃5 min;95 ℃45 s,45-65 ℃45 s,72 ℃1 min,30-40 cycles;72 ℃5-10 min.The amplification products were preserved at 4 ℃.

        The PCR amplification products were examined on a 30% non-denaturing polyacrylamide gel[10]for 30 min-3 h in electrophoresis buffer of 1TBA at a constant voltage of 200-220 V.After the electrophoresis,the gel staining was carried out according to the following procedures:fixation with 10%ethanol and 0.5% acetic acid for 10-15 min,permeating in 0.2% silver nitrate solution for 10-15 min,rinsing twice for 10-30 s with distilled water and staining with 1.5% sodium hydroxide and 0.4%formaldehyde.

        Molecular marker analysis

        Atotal of three types (1:1,1:2:1 and 1:1:1:1) of segregating populations of F2generation of the four-way cross population were marked.The marking results were represented with different lowercase letters.If the band type of the first parent was marked as a,all the same bands were marked as a; the firstly-shown second different band type was marked as b,and all the same bands were marked as b;the firstly-shown third different band type was marked as c,and all the same band were marked as c,and so on.The segregating marking could be divided into four types,back-cross type(aa×ab,aa×ba,ab×aa,ab×bb),F2type (ab×ab,ab×ba),three-way type(ab×cc,aa×bc,ab×ac,ab×ca,ab×bc,ab×cb)and four-way type (ab×cd).In the marking of band types of segregating populations,the homozygous band types were represented as aa,bb,cc and dd,and the heterozygous band types were represented as ab,ac,ad,bc and bd.

        Results and Analysis

        Screening results of SSR primer polymorphism

        The polymorphism screening of the four parents of sweet maize was conducted using SSR primer.As shown in Fig.1,from left to right,every four adjacent bands were divided into one group.The products in each group were amplified using the same SSR primer pair.After the DNA extracted from leaf powder was amplified,clear DNA bands were obtained for SSR primer polymorphism analysis.As shown in Fig.1,the SSR primers were bnlg2086,umc1917,umc1603,umc-1590,bnlg615,bnlg1564,um1261,umc1961,bnlg1621,umc1185,umc-1465 and umc1555,respectively(from left to right).

        Isolating results of populations using SSR molecular marker

        The DNA of the four-way cross population was amplified by bnlg2046.Aseries of clear DNA bands were obtained(Fig.2).

        Discussion

        The rapid and batch-oriented crushing technology of maize leaves will accelerate DNA extraction,and the rapid crushing of maize leaves is also an important part of molecular markerassisted breeding.Currently,the DNA extraction kit has been developed continuously.However,none convenient and rapid crushing program has been provided.For DNA extraction with kit,well-crushed plant sample is the premise.Therefore,the establishment of rapid and batch-oriented crushing method of leaves plays an important role in numerous molecular marker tests.Compared with current technologies,the developed crushing method has following advantages:①Some leaf samples can be crushed simultaneously,and the number of simultaneously crushed leaf samples is related to volume of antifreeze container.②The developed crushing method consumes less time and effort.③The developed crushing method is safe and non-toxic,and does not require toxic biological antioxidants (i.e.β-mercaptoethanol).The operation is also simple.④The DNA bands amplified by SSR primer are clearer,and they fully meet test requirements.

        The results suggest that this method can be applied in different molecular marker-assisted breeding works.The developed method has simple operation,and it is also characterized by rapidness,bulk,low cost and non-toxicity.In a word,the developed rapid and batch-oriented crushing method of leaves is feasible in laboratory tests.

        [1]LEI KR(雷開榮),SHI CY(石春焱),LI MS(李明順),et al.A new method of genomic DNA extraction of maize (一種玉米葉片基因組DNA 快速提取新方法的初步研究)[J].Acta Agriculturae Boreali-Sinica (華北農(nóng)學報),2006,21 (2):10-12.

        [2]ZHOU ZH (周仲華),WANG F (王峰),CHEN JG (陳金湘).From genomics to proteomics:great molecular progress in cotton (Gossypium hirsutum L.)(從基因組學到蛋白質(zhì)組學:棉花分子生物學研究進展)[J].Cotton Science (棉花學報),2012,24(4):370-378.

        [3]JIA M (賈敏).Brief analysis on application of molecular biological technique in modern medicine(淺析分子生物技術(shù)在現(xiàn)在醫(yī)學中的應(yīng)用)[J].BioTech World(生物技術(shù)世界),2014,4:114.

        [4]REINISCH AJ,DONG JM,BRUBAKER CL,et al.A detailed RFLP map of cotton Gossypium hirsutum× Gossypium barbadense:chromosome organization and evolution in a disomic polyploidy genome [J].Genetics,1994,138 (3):829-847.

        [5]ZHANG J,GUO W,ZHANG T.Molecular linkage map of allotetraploid cotton(Gossypium hirsutum L.×Gossypium barbadense L.) with a haploid population[J].Theoretical and Applied Genetics,2002,105(8):1166-1174.

        [6]ULLOA M,MEREDITH WRJR,SHAPPLEY ZW,et al.RFLP genetic linkage maps from four F2:3populations and a joinmap of Gossypium hirsutum L.[J].Theoretical and Applied Genetics,2002,104(2/3):200-208.

        [7]WANG AY (王愛云),LI G (李構(gòu)).Advances on plant cell engineering technology application in rapeseed breeding(植物細胞工程技術(shù)在油菜育種中的應(yīng)用現(xiàn)狀與進展)[J].Chinese Agricultural Science Bulletin (中國農(nóng)學通報),2005,21(4):64-69.

        [8]YIN LP (印麗萍),WU SM (吳申懋),FU YN (傅怡寧),et al.Primer pair,probe and nucleic acid composition for detecting false sorghum and detection method:China (用于檢測假高梁的引物對、探針、核酸組合物及檢測方法:中國),201410462473.3[P].2014-12-03.

        [9]ZHANG Q (張秋).Genetic map construction and QTL mappings of important agronomic traits in common wheat(普通小麥遺傳圖譜構(gòu)建及重要農(nóng)藝性狀 的QTL 定 位)[D].Tai’an:Shandong Agricultural University (泰安:山東農(nóng)業(yè)大學),2012.

        [10]LIU PF(劉鵬飛).QTL mapping of density tolerance and related traits based on four-way cross population in maize(Zea mays L.)(基于四交群體的玉米耐密性及相關(guān)性狀QTL 定位與分析)[D].Lanzhou:Gansu Agricultural University(蘭州:甘肅農(nóng)業(yè)大學),2013.

        猜你喜歡
        農(nóng)學淺析棉花
        中國古代農(nóng)學風土論的形成、演變與價值
        棉花是花嗎?
        蒲松齡《農(nóng)桑經(jīng)》的農(nóng)學思想及其當代啟示
        《廣西農(nóng)學報》投稿指南
        棉花
        小讀者(2020年4期)2020-06-16 03:33:54
        淺析VLAN間靈活互訪
        電子制作(2019年14期)2019-08-20 05:43:30
        淺析35kV隔離開關(guān)常見缺陷及處理
        電子制作(2018年18期)2018-11-14 01:48:26
        淺析“譙”字“酷烈”義
        農(nóng)學
        新校長(2016年5期)2016-02-26 09:29:00
        心中的“棉花糖”
        美丽的小蜜桃在线观看| 日本一二三区在线视频观看| 东风日产车是不是国产的 | 女人被狂躁c到高潮视频| 无套内谢孕妇毛片免费看看| 日韩人妻无码中文字幕一区| 侵犯了美丽丰满人妻中文字幕| 奇米影视色777四色在线首页| 国内揄拍国内精品人妻浪潮av| 欧美日韩一线| 国产一区二区三区精品乱码不卡| 久久久久久久亚洲av无码| 熟妇人妻av无码一区二区三区| 白丝美女被狂躁免费视频网站| 中文字幕熟女激情50路| 永久天堂网av手机版| 激情久久av一区av二区av三区| 中文字幕乱码人妻无码久久久1| 亚洲国产一区二区网站| 日韩av无码精品一二三区| 国产精品 视频一区 二区三区| 亚洲国产av剧一区二区三区| 亚洲精品久久区二区三区蜜桃臀| 免费网站看v片在线18禁无码| 精品久久久久一区二区国产| 日韩精品成人一区二区三区| 美女扒开大腿让男人桶| 久久久久国产一区二区三区| 深夜福利国产| 亚洲国产精品婷婷久久| 欧美俄罗斯40老熟妇| 午夜亚洲AV成人无码国产| 亚洲一区二区岛国高清| 亚洲人成电影网站色| 99久久久无码国产aaa精品| 国产精品欧美久久久久久日本一道| 人人爽久久涩噜噜噜av| 国产欧美另类精品久久久| 三级网站亚洲三级一区| 樱桃视频影院在线播放| 香蕉视频毛片|