利用CODEHOP设计简并引物并克隆玫瑰LAZY1基因片段
|
摘要
为克隆玫瑰LAZY1基因,介绍使用CODEHOP设计简并引物的方法。本文使用CODEHOP设计出10条简并引物,以玫瑰幼茎提取的RNA反转录所得到的cDNA为模板,经RT-PCR扩增得到长度为410bp的基因片段,将该产物连接到pMD-18T载体中,转化至Trans1-T1大肠杆菌中进行测序,测序片段在Genbank中通过Blastx检索与其他物种LAZY1基因的同源性,发现产物片段与其他植物中的LAZY1基因具有较高的相似性和同源性。研究结果表明CODEHOP在线设计简并引物方法简单实用,且具有较高阳性率。
In order to clone Rosa rugosa LAZY1 gene,introduce the use of CODEHOP which could design degenerate primers.This study designed 10 degenerate primers by CODEHOP,and obtained 410 bp fragment by RT-PCR amplification using cDNA obtained from the reverse transcription of RNA extracted from the stem of rose. The fragment was connected to pMD-18 T vector and transformed into Trans1-T1 Escherichia coli for sequencing. The sequenced fragments were identified by Blastx in Genbank and compared with other LAZY1 genes. It was found that the product fragment had high similarity and homology with other LAZY1 genes in other plants. The results showed that using CODEHOP to design degenerate primers was simple, practical and had high positive rate.
In order to clone Rosa rugosa LAZY1 gene,introduce the use of CODEHOP which could design degenerate primers.This study designed 10 degenerate primers by CODEHOP,and obtained 410 bp fragment by RT-PCR amplification using cDNA obtained from the reverse transcription of RNA extracted from the stem of rose. The fragment was connected to pMD-18 T vector and transformed into Trans1-T1 Escherichia coli for sequencing. The sequenced fragments were identified by Blastx in Genbank and compared with other LAZY1 genes. It was found that the product fragment had high similarity and homology with other LAZY1 genes in other plants. The results showed that using CODEHOP to design degenerate primers was simple, practical and had high positive rate.
引文
[1]徐宗大,赵兰勇,张玲.玫瑰SRAP遗传多样性分析与品种指纹图谱构建[J].中国农业科学,2011,44(8):1662-1669.
[2]张淑梅.地被菊匍匐生长特性形成机理研究[D].南京农业大学,2008.
[3]张磊,叶志玮.沙柳Sps LAZY1a和Sps LAZY1b基因克隆及生物信息学分析[J].西北林学院学报,2017,32(1):98-105.
[4]Chris D,Ann C.Ppe TAC1 promotes the horizontal growth of branches in peach trees and is a member of a functionally conserved gene family found in diverse plants species[J].The Plant Journal,2013,75:618–630.
[5]Jessica M,Guseman.DRO1 influences root system architecture in Arabidopsis and Prunus species[J].The Plant Journal,2017,89:1093-1105.
[6]Baisheng Yu,Zhongwei Lin,Haixia Li,et al.TAC1,a major quantitative trait locus controlling tiller angle in rice[J].The Plant Journal,2007,52:891-898.
[7]Takeshi Y,Edgar P.At LAZY1 is a signaling component required for gravitropism of the Arabidopsis thaliana inflorescence[J].The Plant Journal,2013,74:267-279.
[8]姜川.玉米负重力性生长相关基因LAZY1的克隆及功能分析[D].山东农业大学,2012.
[9]王少峡,陈丽媛,张竞秋.利用生物信息学资源设计简并引物[J].天津师范大学学报,2006,26(2).
[10]李玉恒,赵明慧,赵紫阳.利用梯度PCR和降落PCR扩增CIRP基因的比较[J].黑龙江八一农垦大学学报,2011,23(5):46-49.
[11]万兵,闫杰.一种优化的PCR方法—降落PCR扩增目的基因[J].江苏临床医学杂志,2002,6(2).
[12]田路明,黄丛林.降落PCR法快速检测高羊茅转基因植株[J].生物技术通报,2006,3,023.
[13]张瑞强,繁萍.利用降落PCR扩增KMT-1基因[J].青海大学学报,2010,28(2).
[14]马月林.沙柳TAC1基因克隆与生物信息学及组织表达特性分析[D].内蒙古农业大学,2016.
[15]佘朝文,蒋向辉.CODEHOP在线简并引物设计与杉木4CL基因片段克隆[J].湖南临沂科技,2009,36(6)..
[16]夏志强,陈新,王海燕.简并引物设计与大戟科3中植物WAX基因片段的克隆[J].安徽农学通报,2012,23.034.
[17]李运合,孙光明.利用CODEHOP和i CODEHOP设计简并引物克隆芒果LAX基因家族片段[J].热带作物学报,2011,32(12):2278-2282.
[18]齐育平,周月婷.利用CODEHOP设计简并引物克隆红色红曲霉丝氨酸羧肽酶基因片段和序列分析[J].浙江师范大学学报,2014,37(1).
[2]张淑梅.地被菊匍匐生长特性形成机理研究[D].南京农业大学,2008.
[3]张磊,叶志玮.沙柳Sps LAZY1a和Sps LAZY1b基因克隆及生物信息学分析[J].西北林学院学报,2017,32(1):98-105.
[4]Chris D,Ann C.Ppe TAC1 promotes the horizontal growth of branches in peach trees and is a member of a functionally conserved gene family found in diverse plants species[J].The Plant Journal,2013,75:618–630.
[5]Jessica M,Guseman.DRO1 influences root system architecture in Arabidopsis and Prunus species[J].The Plant Journal,2017,89:1093-1105.
[6]Baisheng Yu,Zhongwei Lin,Haixia Li,et al.TAC1,a major quantitative trait locus controlling tiller angle in rice[J].The Plant Journal,2007,52:891-898.
[7]Takeshi Y,Edgar P.At LAZY1 is a signaling component required for gravitropism of the Arabidopsis thaliana inflorescence[J].The Plant Journal,2013,74:267-279.
[8]姜川.玉米负重力性生长相关基因LAZY1的克隆及功能分析[D].山东农业大学,2012.
[9]王少峡,陈丽媛,张竞秋.利用生物信息学资源设计简并引物[J].天津师范大学学报,2006,26(2).
[10]李玉恒,赵明慧,赵紫阳.利用梯度PCR和降落PCR扩增CIRP基因的比较[J].黑龙江八一农垦大学学报,2011,23(5):46-49.
[11]万兵,闫杰.一种优化的PCR方法—降落PCR扩增目的基因[J].江苏临床医学杂志,2002,6(2).
[12]田路明,黄丛林.降落PCR法快速检测高羊茅转基因植株[J].生物技术通报,2006,3,023.
[13]张瑞强,繁萍.利用降落PCR扩增KMT-1基因[J].青海大学学报,2010,28(2).
[14]马月林.沙柳TAC1基因克隆与生物信息学及组织表达特性分析[D].内蒙古农业大学,2016.
[15]佘朝文,蒋向辉.CODEHOP在线简并引物设计与杉木4CL基因片段克隆[J].湖南临沂科技,2009,36(6)..
[16]夏志强,陈新,王海燕.简并引物设计与大戟科3中植物WAX基因片段的克隆[J].安徽农学通报,2012,23.034.
[17]李运合,孙光明.利用CODEHOP和i CODEHOP设计简并引物克隆芒果LAX基因家族片段[J].热带作物学报,2011,32(12):2278-2282.
[18]齐育平,周月婷.利用CODEHOP设计简并引物克隆红色红曲霉丝氨酸羧肽酶基因片段和序列分析[J].浙江师范大学学报,2014,37(1).