CN102206655A - 茄子花青素合成二氢黄酮醇4-还原酶的基因序列 - Google Patents
茄子花青素合成二氢黄酮醇4-还原酶的基因序列 Download PDFInfo
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Abstract
本发明为生物技术领域的一种茄子花青素合成二氢黄酮醇4-还原酶的基因序列，该序列具有SEQ ID NO.1中从核苷酸第1-1149位所示的核苷酸序列；另一种序列具有SEQ ID NO.3中从核苷酸第1-1149位所示的核苷酸序列。本发明可获得在高花青素、抗水分胁迫、抗紫外保护等方面具有重要特性的茄子品种；利用本发明的茄子二氢黄酮醇4-还原酶基因，通过各种常规筛选方法，可筛选出与二氢黄酮醇4-还原酶相关发生相互作用的物质、受体、抑制剂或拮抗剂等。
Description
技术领域
本发明涉及的是一种生物技术领域的基因序列，具体是一种茄子花青素合成二氢黄酮醇4-还原酶的基因序列。
背景技术
花青素是自然界仅次于叶绿素的植物体第二大色素类型，属于类黄酮类物质。花青素是一种水溶性色素，偏酸性，被贮藏在植物细胞的液泡中。植物的许多器官和组织的颜色都是由花青素决定的。近几十年来，作为多酚类物质的花青素的保健作用开始被深入研究，花青素所具有的抗氧化，自由基清除等作用，越来越受到人们的重视。茄子，尤其是紫色茄子，是为数不多的富含花青素的蔬菜种类，营养价值丰富。
Davies等在《Euphytica》2003年第三卷第259-268页发表的《 Enhancing anthocyaninproduction by altering competition for substrate between flavonol synthase and
与同为茄属的马铃薯和番茄的深入研究相比，茄子的花青素生物合成的研究非常滞后。如今，提高植物体内类黄酮(花青素)类物质已经成为了植物育种的一个重要的方向。而育种的基础就是完全理解花青素合成和调节的因子以及相关酶类的作用。茄子作为一种重要的蔬菜作物，对其花青素合成路径的研究将会对于高花青素茄子品种的育种，以及茄子的抗氧化，保健作用的进一步开发提供理论基础。
经对现有技术文献的检索，尚未发现与本发明相同或者相似的技术主题。
发明内容
本发明的目的在于克服现有技术中的不足，公开了茄子花青素合成二氢黄酮醇4-还原酶的基因序列。利用本发明的茄子二氢黄酮醇4-还原酶，可筛选出与茄子二氢黄酮醇4-还原酶相关发生相互作用的物质、受体、抑制剂或拮抗剂等，利用本发明可获得在高花青素、抗紫外保护、抗水分胁迫等方面具有重要特性的茄子品种。
本发明是通过以下技术方案实现的，
本发明涉及一种茄子花青素合成二氢黄酮醇4-还原酶的基因序列，该序列具有SEQ IDNO.1中从核苷酸第1-1149位所示的核苷酸序列。
所述的序列编码的多肽具有SEQ ID NO.2所示的氨基酸序列。
本发明还涉及一种茄子花青素合成二氢黄酮醇4-还原酶的基因序列，该序列具有SEQ IDNO.3中从核苷酸第1-1149位所示的核苷酸序列。
所述的序列编码的多肽具有SEQ ID NO.4所示的氨基酸序列。
本发明根据茄子基因序列数据库，运用生物信息学手段，通过序列比较和电子克隆拼接，设计出合适的引物，用分子克隆的方法，克隆出紫色茄子和白色茄子中二氢黄酮醇4-还原酶(SmDFR)基因；经过功能鉴定，确定分离出的是二氢黄酮醇4-还原酶基因，紫色茄子和白色茄子中，SmDFR基因序列有两个碱基差异，氨基酸序列有一个氨基酸残基的差异。并且证明了SmDFR可能是造成茄子花青素差异的关键基因。
本发明具有如下的有益效果：利用本发明的茄子二氢黄酮醇4-还原酶，可筛选出与茄子二氢黄酮醇4-还原酶相关发生相互作用的物质、受体、抑制剂或拮抗剂等，利用本发明可获得在高花青素、抗紫外保护、抗水分胁迫等方面具有重要特性的茄子品种。
本发明涉及的大肠杆菌DH5α，BL-21菌株已在《萨姆布鲁克J，拉塞尔D W.分子克隆实验指南[M].黄培堂，王嘉玺，朱厚础，等译.第3版.北京：科学出版社，2002》中公开；大肠杆菌DH5α，BL-21可通过公开市售的商业渠道取得，如上海天根生物公司，公司地址：上海市漕溪路258弄27号航星商务楼1号楼606室。
具体实施方式
下面结合具体的实施例子，进一步阐述本发明的具体实施方式。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等(1989)编著的分子克隆实验手册(New York：Cold SpringHarbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。实验所用的PrimeScriptTM 1st Strand cDNA Synthesis Kit、高保真的Taq酶、pMD19-T载体是大连TaKaRa公司产品。
实施例1
紫色茄子二氢黄酮醇4-还原酶(YLP SmDFR)的克隆、表达和鉴定
步骤一、紫色茄子SmDFR二氢黄酮醇4-还原酶基因的克隆
1、茄子实验材料取材
以本实验室收集和保存茄子种质资源云南紫长茄(下简称紫长茄，英文简称YLP)为实验材料。紫长茄为具有代表性的紫色茄子资源，取材于云南省玉溪市，具有紫色茄子栽培品种所共有的无刺，紫茎，紫花表型。商品成熟期的果实，黑紫色，果型长而大，故名紫长茄。实验材料栽培于上海市闵行区浦江镇航天育种基地的人工塑料大棚中。在全自然条件下育苗，生长，并结实。实验取材需用到紫长茄的紫色茎，紫色花瓣，绿色叶片，未成熟期果实的白色果皮，商品成熟期果实的黑色果皮和生理成熟期的黄色果皮。
2、TRIzol法提取商品成熟期紫色果皮中的总RNA
(1)称取0.5g紫长茄的商品成熟期紫色果皮，用液氮速冻后，迅速研磨成细粉，分装于1.5ml的eppendorf离心管中；
(2)加入1ml TRIzol，用力摇动使混合均匀，25℃，放置5min；
(3)每管加0.2ml氯仿，用力振荡15sec，室温放置2min-3min，4℃，12,000g，离心15min；
(4)将上清液600ul吸入1.5ml的新eppendorf离心管中，加与上清等体积异丙醇，颠倒混匀，-20℃冰箱中放置30min；
(5)4℃，12,000g，离心15min，弃上清，收集RNA沉淀，加1ml 75％(V/V)乙醇洗涤沉淀1-2次，洗涤具体为：加1ml 75％(V/V)乙醇，4℃离心，12000g，10min-15min，弃上清，收集RNA沉淀；
(6)沉淀25℃下干燥15min-20min，溶于30μl-50μl的RNase-free water中；
(7)用甲醛变性胶电泳鉴定总RNA质量，然后在分光光度计上测定RNA含量。
3、YLP SmDFR基因cDNA的克隆
基于Genbank中茄子的推定SmDFR基因部分片段序列设计RACE引物(表1)，扩增两个序列的5’端和3’端全长序列。RACE实验使用SMARTerTM RACE cDNA Amplification Kit试剂盒(Takara)进行操作。经过巢式PCR检测后，将PCR扩增产物克隆至pMD-19T载体(Takara)，导入大肠杆菌DH5α菌株。通过3’RACE和5’RACE实验，克隆了紫长茄的SmDFR基因的cDNA全长序列。cDNA长度为1503bp，蛋白编码区从87-1235共1149bp，推导编码383个氨基酸，cDNA编码区全序列和推导蛋白序列见SEQ ID.NO.1和SEQ ID.NO.2。
表1：SmDFR的RACE扩增引物序列
将测序得到的核苷酸序列用NCBI的BLASTn方法与Genbank中已登录的DFR基因序列进行比对，与同为茄科的马铃薯的DFR基因的编码区序列的同源度最高，具体见表2。大肠杆菌原核表达证实SEQ ID NO.1是茄子二氢黄酮醇4-还原酶基因，与已报道的马铃薯(Solanum tuberosum)同源度为94.8％，烟草(Nicotiana tabacum)为90.8％，矮牵牛(Petuniax hybrida)为89％，胡萝卜(Daucus carota)为66.8％，拟南芥(Arabidopsis thalina)为64.2％。
表2 YLP SmDFR紫色茄子二氢黄酮醇4-还原酶与马铃薯StDFR的核苷酸序列的同源比较表
StDFR
ATGGCAAGTGAAGTTCATTCAGTTGTTGATGCCCATTCTCCCCCGAAGACGCCAACG
GTT
SmDFR
ATGGCAAGTGAAGCTCATGCAGTTGTTGATGCCCCTTCTCCTCCGGAGGCACCGAC
GGTT
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR
TGCGTCACAGGAGCAGCTGGATTTATCGGCTCTTGGCTTGTCATGAGACTCCTTGAA
CGC
SmDFR
TGCGTCACAGGAGCAGCTGGATTTATTGGCTCTTGGCTTGTCATGAGACTCCTTGAA
CGC
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR
GGTTATAATGTCCATGCTACTGTTCGTGATCCTGAGAACCAGAAGAAGGTGAAACAT
CTG
SmDFR
GGTTATAATGTCCATGCTACTGTTCGTGATCCTGAGAACAAGAAGAAGGTGAAACAT
CTG
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR
TTGGAACTGCCAAAAGCTGATACAAACTTAACGCTGTGGAAAGCAGACTTGGCAGT
GGAA
SmDFR
TTGGAATTGCCTAAAGCTGATACAAACTTAACGCTGTGGAAAGCAGACTTGAATGTG
GAA
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR
GGAAGCTTTGATGAAGCCATTCAAGGCTGTCAAGGAGTATTTCATGTGGCTACACCG
ATG
SmDFR
GGAAGCTTTGATGAAGCCATTCAAGGCTGTCAGGGAGTATTTCATGTGGCTACACCG
ATG
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR
GATTTCGAGTCCAAGGATCCAGAGAACGAAGTAATTAAACCAACAGTCAGGGGAAT
GCTA
SmDFR
GATTTCGAGTCCAAGGACCCTGAGAATGGAGTAATTAAACCAACAGTCAGGGGAAT
GTTG
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR
AGTATCATAGAATCATGTGCTAAAGCTAACACAGTGAAGAGGCTGGTTTTCACTTCAT
CT
SmDFR
AGCATCATAGAATCATGTGCTAAAGCTAACACAGTAAAGAGGCTGGTTTTCACTTCAT
CT
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR
GCTGGAACTCTTGATGTCCAAGAGGACCAAAAACTCTTCTATGACGAGACCAGCTG
GAGC
SmDFR
GCTGGAACTCTTGATGTCCAAGAGAACCAGAAACTCTTCTATGACGAGACTAGCTGG
AGC
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR
GATTTGGACTTCATATATGCTAAGAAGATGACAGGATGGATGTATTTTGTTTCCAAGA
TA
SmDFR
GACTTGGACTTCATATATGCTAAGAAGATGACAGGATGGATGTATTTTGTTTCCAAGA
TA
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR
CTGGCAGAGAAGGCTGCAATGAAAGAAGCTAAAAAGAATAACATTAATTTCATTAGC
ATC
SmDFR
CTGGCAGAGAAGGCTGCAAGGGAAGAAGCTAAAAAAAATAACATTGATTTCATTAGC
ATC
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR
ATACCACCACTGGTTGTTGGTCCATTCATCACACCTACGTTCCCACCTAGCTTAATCA
CT
SmDFR
ATACCACCACTGGTTGTTGGTCCATTCATCACACCTACGTTTCCACCTAGCTTAATCA
CT
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR
GCCCTTTCACTAATTACCGGGAATGAAGCTCACTACGGCATCATTAAACAAGGTCAAT
AT
SmDFR
GCCCTTTCAGTAATTACCGGGAATGAAGCTCACTACTGCATCATTAAACAAGGTCAAT
AT
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR
GTGCATTTGGATGATCTTTGTGAGGCTCATATATTCCTGTATGAGCACCCCAAGGCA
GAG
SmDFR
GTGCATCTGGATGATCTTTGTGAGGCTCATATATTCCTGTATGAGCACCCCAAGGCA
GAG
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR
GGAAGATTCATTTGCTCGTCCCATCATGCTATCATCTACGATGTGGCTAAGATGGTGC
GA
SmDFR
GGAAGATTCATTTGCTCATCCCATCATGCTATCATCTACGATGTGGCTAAGATGGTGC
GA
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR
CAGAAATGGCCAGAGTACTATGTTCCTACTGAGTTTAAGGGTATCGATAAGGACTTG
CCC
SmDFR
CAGAAATGGCCAGATTACTATGTTCCTACTGAGTTTAAAGGTATCGACAAGGAATTG
CCC
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR
ATAGTGTCTTTTTCATCAAAGAAGCTTATGGACATGGGGTTTCAATTCAAATACACTT
TG
SmDFR
ATAGTGTCTTTTTCATCAAAGAGGCTTACGGATATGGGGTTTGAATTCAAATACACTC
TG
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR
GAGGATATGTATAAAGGGGCCATTGAGACTTGCCGACAGAAGCAGTTGCTTCCCTTT
TCT
SmDFR
GAGGATATGTATCAAGGGGCCATTGAGACTTGCCGACAGAAGCAGTTGCTTCCCTTT
TCT
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR
ACCCGAAGCTCTGCAGACAATGGAAAAGACAAAGAAGCAATTCCCATTTCTACTGAA
AAC
SmDFR
ACCCGAAGCACTCTAGACAATGGAGAAGATAAAGAAACCATTCCCATATCTACTGAA
AAC
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR
TATTCAAGTGGCAAGGAGAATGCACCAGTTGCCAATTGTACAGGAAAGTTTACCAAT
GGT
SmDFR
TATGCAAGTGGCAAGGAGAATGCACCAGTTGCAAATAGTACAGGACAGTTGACCAAT
GGT
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
StDFR GAAATCTAG
SmDFR GAAATCTAG
＊＊＊＊＊＊＊＊＊
SmDFR：茄子二氢黄酮醇4-还原酶核苷酸序列；
StDFR：马铃薯StDFR的核苷酸序列(AY289922)；
步骤二、紫色茄子SmDFR基因的表达与拷贝数分析
1.YLP SmDFR基因的荧光定量PCR分析
取紫长茄的紫色茎，叶片，紫色花瓣，未成熟果实白色果皮，商品成熟期果实的紫色果皮，生理成熟期果实黄色果皮的总RNA，采用SmDFR-F2和SmDFR-R2引物对进行SmDFR基因的荧光定量PCR分析：
SmDFR-F2为上游引物，引物序列为AAGGCTGTCAGGGAGTATTTCA，
SmDFR-R2为下游引物，引物序列为AGTTTCTGGTTCTCTTGGACATC。
以SmACTIN基因作为内参对照，采用SmACTIN-F1和SmACTIN-R1引物对：
SmACTIN-F1为上游引物，引物序列为TTCCTTGTATGCTAGTGGTCGTACAA，
SmACTIN-R1为下游引物，引物序列为：CTCAGCACCAATGGTAATAACTTGTCC。
标准曲线采用克隆到pMD-19T载体上的DFR部分片段和ACTIN部分片段的质粒DNA绘制，设置5个标准样品，分别是质粒DNA原液和质粒DNA稀释10倍，100倍，1000倍和10000倍的DNA样品。定量PCR的操作采用SYBR PrimeScript RT-PCR Kit(Takara)，实验步骤按照试剂盒说明书进行。荧光定量PCR反应在Mastercycler ep realplex(Eppendorf)PCR仪上进行。反应条件为预变性94℃2min；94℃20s，52℃20s，72℃30s，35个循环；72℃1min。
实验结果表明，SmDFR在紫长茄的紫色组织，即紫茎，紫色花瓣，商品成熟期紫色果实果皮中的表达量均显著高于非紫色组织，即叶片，未成熟期白色果实果皮，生理成熟期黄色果实果皮。
2.YLP SmDFR基因的拷贝数分析
为了确定YLP SmDFR基因的拷贝数，提取紫长茄的基因组DNA，进行Southern杂交试验。提取DNA的具体方法如下：
取紫长茄的幼嫩叶片，采用CTAB方法提取。具体步骤如下：
(1)取0.3g新鲜幼嫩的叶片在液氮中快速研磨成粉末后，置于2ml的离心管中；加入500μlCTAB提取缓冲液，60℃水浴45min；CTAB提取缓冲液的配方为：CTAB 4g，氯化钠16.4g，Tris-HCl 20ml(pH 8.0)，0.5M EDTA 8ml，用去离子水70ml溶解后，定容到200ml，高压灭菌，冷却后加入0.5％的巯基乙醇(v/v)。
(2)待冷却后，加入500μl苯酚-氯仿-异戊醇(分析纯液体，25∶24∶1体积比)抽提混合液，混匀后，12000r/m离心10min；
(3)上清液转入新管，加入500μl氯仿-异戊醇(分析纯液体，24∶1体积比)抽提混合液，混匀后，12000r/m离心10min；取上清液转入新管，加入500ul冷处理的异丙醇，轻轻混匀，冰浴30min，4℃，8000r/m离心10min；
(4)倒去上清液，加入去离子水300ul溶解后，加入150ul，7.5mol/L NH4Ac混匀，冰浴20min，4℃，10000r/m离心20min；
(5)取上清液，加入预冷的无水乙醇900ul，冰浴20min，4℃，8000r/m离心20min；倒去上清液，70％乙醇冲洗两次，干燥后，加入去离子水150ul溶解后，加入RNase除去RNA，37℃水浴30min。DNA提取后，在分光光度计上测定DNA含量。
DNA探针采用回收纯化的SmDFR的cDNA的扩增片段，片段回收方法为将扩增SmDFR的cDNA全长序列电泳，紫外灯下割取DNA条带，用AxyPrepTM DNA gel Extraction Kit试剂盒回收，回收方法参照试剂盒说明书。使用DIG-High Prime DNA Labeling and DetectionStarter Kit I(Roche)试剂盒进行探针标记。标记方法参见试剂盒说明书。
采用
BamH I内切酶(Fermentas)进行基因组DNA的酶切。酶切体系参照内切酶说明书，在37℃水浴条件下，酶切6h。酶切后的DNA在20V电压下进行0.8％浓度琼脂糖电泳10h。电泳完毕后的琼脂糖凝胶采用进行脱嘌呤，变性和中和操作。将凝胶浸入100ml脱嘌呤液(0.25N的盐酸)中，15min，然后浸入100ml变性液(0.5mol/LNaOH，1.5mol/L氯化钠)30min，再浸入100ml中和液(0.5mol/L Tris-HCl，pH7.5，1.5mol/L氯化钠)30min。
凝胶浸入20×SSC液体中，采用VACUGENE XL BLOTTING UNIT真空转印系统(GE)将DNA转移到HYBOND-N+尼龙膜上。转印后的尼龙膜用2×SSC洗涤片刻后，120℃的烘箱内烘烤30min进行DNA交联。探针杂交和显色步骤也按照DIG-High Prime DNA Labeling andDetection Starter Kit I试剂盒说明书进行。20×SSC溶液配方为：氯化钠175.3g，柠檬酸钠88.2g用离子水定容到1L，pH7.0。2×SSC溶液为20×SSC溶液用去离子水稀释10倍。
DNA印迹结果显示，SmDFR在紫长茄中为单拷贝基因。
步骤三、紫色茄子SmDFR酶的功能验证
1.YLP SmDFR原核表达载体的构建
YLP SmDFR基因的原核表达采用了pMAL-c2x载体(New England BioLabs)。载体采用BamHI和SalI双酶切，采用
BamH I内切酶(Fermentas)进行基酶切操作。酶切体系参照内切酶说明书，在37℃水浴条件下，酶切15min。
DFR的ORF区域采用带有BamHI酶切位点的5’端引物和带有XhoI酶切位点的3’端引物扩增，引物序列为：
上游引物：CGCGGATCCATGGCAAGTGAAGCTC，
下游引物：ATTCTCGAGCTAGATTTCACCATTGGTCAA。
PCR扩增DFR的ORF区域，每个循环内72℃延伸时间为90s，退火温度为60℃。扩增条带用BamHI和XhoI双酶切，酶切采用
BamH I内切酶(Fermentas)进行基酶切操作。酶切体系参照内切酶说明书，在37℃水浴条件下，酶切15min。回收步骤将扩增片段电泳，紫外灯下割取DNA条带，用AxyPrepTM DNA gel Extraction Kit试剂盒回收，回收方法参照试剂盒说明书。
酶切过的DNA和载体采用DNA Ligation Kit Ver.2.0试剂盒(Takara)连接，连接方法参见试剂盒说明书。热击转化入大肠杆菌BL-21感受态菌株。在有氨苄的LB固体平板培养基上37℃培养过夜，挑取单菌落，测序确认连接状况。LB培养基的配方为：配制每升培养基，用950ml去离子水中加入10g胰化蛋白胨，5g酵母提取物，10g氯化钠。调节pH至7.0，定容到1L体积。15psi高压下蒸汽灭菌20min。LB固体培养基配方为在LB液体培养基中，加入15g琼脂粉。15psi高压下蒸汽灭菌20min。
2.YLP SmDFR的融合蛋白诱导
挑选生长状态良好的BL-21菌株单克隆，转接到1L的LB液体培养基的三角瓶内，其中含有氨苄50μg/ml。37℃，200-225rpm摇菌4h左右，使OD600为1.0左右，将培养物转入20-22℃的摇床中以200-250rpm振摇1小时后加1ml 1M IPTG(异丙基-β-D-硫代吡喃半乳糖苷)(终浓度为1mM)，在20℃条件下以80-100rpm振摇过夜。随后10000rpm，4℃，离心10分钟后，收集大肠杆菌细胞。将沉淀物重悬于50ml裂解溶液中，并在冰上冷却10分钟。在冰浴条件下，使用超声细胞破碎仪破碎细胞。随后将破速的细胞在15000rpm条件下离心30min，取上清。在上清中加入1ml用裂解溶液清洗并平衡的直链淀粉微珠(Amylose resin)(NewEngland Biolabs)，同时再向溶液加入另50ml裂解溶液并平均分为两部分，4℃孵育过夜。之后1000rpm，4℃条件下慢速离心1min后，弃上清。用50ml清洗溶液清洗微珠三次后，用1ml洗脱溶液洗脱。
其中菌体裂解溶液的配方为：20mM Tris-HCl，pH8.0，200mM氯化钠，1mM PMSF，EDTA-free的蛋白酶抑制剂(Promega)，10％甘油；清洗溶液的配方为：20mM Tris-HCl，pH8.0，200mM氯化钠，10％甘油；洗脱溶液的配方为：20mM Tris-HCl，pH8.0，200mM氯化钠，50mM麦芽糖，10％甘油。
结果表明，SmDFR的融合蛋白对于三种反应底物均具有活性。能够将二氢山奈酚，二氢檞皮素，二氢杨梅素转化为相应的底物。具有二氢黄酮醇4-还原酶的活性。并且对于二氢杨梅素的活性要显著大于二氢山奈酚和二氢檞皮素，表明该酶还具有底物选择性的特定，这符合紫色茄子中主要二氢杨梅素的衍生物，飞燕草素类花青素的事实。
实施例2
白色茄子二氢黄酮醇4-还原酶(YRW SmDFR)的克隆、表达和鉴定
步骤一、白色茄子SmDFR二氢黄酮醇4-还原酶基因的克隆
1、茄子实验材料取材
以本实验室收集和保存茄子种质资源云南圆白茄(下简称圆白茄，英文简称YRW)为实验材料。圆白茄为具有代表性的白色茄子资源，取材于云南省玉溪市，具有白色茄子栽培品种所共有的果实表型，同时还有部分茄子野生品种的表性特征：多刺，绿茎，白花表型。商品成熟期的果实，乳白色，果型大而圆，故名圆白茄。实验材料栽培于上海市闵行区浦江镇航天育种基地的人工塑料大棚中。在全自然条件下育苗，生长，并结实。实验取材需用到圆白茄的绿色茎，白色花瓣，绿色叶片，未成熟期果实的白色果皮，商品成熟期果实的乳白色果皮和生理成熟期的黄色果皮。
2、TRIzol法提取商品成熟期紫色果皮中的总RNA
(1)称取0.5g圆白茄的商品成熟期白色果皮，用液氮速冻后，迅速研磨成细粉，分装于1.5ml的eppendorf离心管中；
(2)加入1ml TRIzol，用力摇动使混合均匀，25℃，放置5min；
(3)每管加0.2ml氯仿，用力振荡15sec，室温放置2min-3min，4℃，12,000g，离心15min；
(4)将上清液600ul吸入1.5ml的新eppendorf离心管中，加与上清等体积异丙醇，颠倒混匀，-20℃冰箱中放置30min；
(5)4℃，12,000g，离心15min，弃上清，收集RNA沉淀，加1ml 75％(V/V)乙醇洗涤沉淀1-2次，洗涤具体为：加1ml 75％(V/V)乙醇，4℃离心，12000g，10min-15min，弃上清，收集RNA沉淀；
(6)沉淀25℃下干燥15min-20min，溶于30μl-50μl的RNase-free water中；
(7)用甲醛变性胶电泳鉴定总RNA质量，然后在分光光度计上测定RNA含量。
3、YRW SmDFR基因cDNA的克隆
基于YLP SmDFR的全长克隆结果，设计全场的上下游引物：
上游引物为5’-ATGGCAAGTGAAGCTCATGCA-3’，
下游引物为5’-CTAGATTTCACCATTGGTCAACT-3’。
克隆的YRW SmDFR的cDNA长度为1503bp，蛋白编码区从87-1235共1149bp，推导编码383个氨基酸，cDNA编码区全序列和推导蛋白序列见SEQ ID.NO.3和SEQ ID.NO.4。
通过序列分析，SEQ ID NO.3与SEQ ID NO.1在632位点和792位点有两个碱基发生了变化(见表3，用小写标出)。SEQ ID.NO.4与SEQ ID NO.2在第236位点有一个氨基酸残基差异。
将测序得到的核苷酸序列用NCBI的BLASTn方法与Genbank中已登录的DFR基因序列进行比对，与同为茄科的马铃薯的DFR基因的编码区序列的同源度最高。大肠杆菌原核表达证实SEQ ID NO.3是茄子二氢黄酮醇4-还原酶基因，与已报道的马铃薯(Solanumtuberosum)同源度为94.8％，烟草(Nicotiana tabacum)为90.8％，矮牵牛(Petunia x hybrida)为89％，胡萝卜(Daucus carota)为66.8％，拟南芥(Arabidopsis thalina)为64.2％。
表3YRW SmDFR白色茄子二氢黄酮醇4-还原酶与YLPSmDFR的核苷酸序列的同源比
较表
YLP
ATGGCAAGTGAAGCTCATGCAGTTGTTGATGCCCCTTCTCCTCCGGAGGCACCGAC
GGTT
YRW
ATGGCAAGTGAAGCTCATGCAGTTGTTGATGCCCCTTCTCCTCCGGAGGCACCGAC
GGTT
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP
TGCGTCACAGGAGCAGCTGGATTTATTGGCTCTTGGCTTGTCATGAGACTCCTTGAA
CGC
YRW
TGCGTCACAGGAGCAGCTGGATTTATTGGCTCTTGGCTTGTCATGAGACTCCTTGAA
CGC
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP
GGTTATAATGTCCATGCTACTGTTCGTGATCCTGAGAACAAGAAGAAGGTGAAACAT
CTG
YRW
GGTTATAATGTCCATGCTACTGTTCGTGATCCTGAGAACAAGAAGAAGGTGAAACAT
CTG
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP
TTGGAATTGCCTAAAGCTGATACAAACTTAACGCTGTGGAAAGCAGACTTGAATGTG
GAA
YRW
TTGGAATTGCCTAAAGCTGATACAAACTTAACGCTGTGGAAAGCAGACTTGAATGTG
GAA
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP
GGAAGCTTTGATGAAGCCATTCAAGGCTGTCAGGGAGTATTTCATGTGGCTACACCG
ATG
YRW
GGAAGCTTTGATGAAGCCATTCAAGGCTGTCAGGGAGTATTTCATGTGGCTACACCG
ATG
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP
GATTTCGAGTCCAAGGACCCTGAGAATGGAGTAATTAAACCAACAGTCAGGGGAAT
GTTG
YRW
GATTTCGAGTCCAAGGACCCTGAGAATGGAGTAATTAAACCAACAGTCAGGGGAAT
GTTG
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP
AGCATCATAGAATCATGTGCTAAAGCTAACACAGTAAAGAGGCTGGTTTTCACTTCAT
CT
YRW
AGCATCATAGAATCATGTGCTAAAGCTAACACAGTAAAGAGGCTGGTTTTCACTTCAT
CT
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP
GCTGGAACTCTTGATGTCCAAGAGAACCAGAAACTCTTCTATGACGAGACTAGCTGG
AGC
YRW
GCTGGAACTCTTGATGTCCAAGAGAACCAGAAACTCTTCTATGACGAGACTAGCTGG
AGC
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP
GACTTGGACTTCATATATGCTAAGAAGATGACAGGATGGATGTATTTTGTTTCCAAGA
TA
YRW
GACTTGGACTTCATATATGCTAAGAAGATGACAGGATGGATGTATTTTGTTTCCAAGA
TA
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP
CTGGCaGAGAAGGCTGCAAGGGAAGAAGCTAAAAAAAATAACATTGATTTCATTAGC
ATC
YRW
CTGGCgGAGAAGGCTGCAAGGGAAGAAGCTAAAAAAAATAACATTGATTTCATTAGC
ATC
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP
ATACCACCACTGGTTGTTGGTCCATTCATCACACCTACGTTTCCACCTAGCTTAATCA
CT
YRW
ATACCACCACTGGTTGTTGGTCCATTCATCACACCTACGTTTCCACCTAGCTTAATCA
CT
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP
GCCCTTTCAGTAATTACCGGGAATGAAGCTCACTACTGCATCATTaAACAAGGTCAAT
AT
YRW
GCCCTTTCAGTAATTACCGGGAATGAAGCTCACTACTGCATCATTgAACAAGGTCAAT
AT
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP
GTGCATCTGGATGATCTTTGTGAGGCTCATATATTCCTGTATGAGCACCCCAAGGCA
GAG
YRW
GTGCATCTGGATGATCTTTGTGAGGCTCATATATTCCTGTATGAGCACCCCAAGGCA
GAG
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP
GGAAGATTCATTTGCTCATCCCATCATGCTATCATCTACGATGTGGCTAAGATGGTGC
GA
YRW
GGAAGATTCATTTGCTCATCCCATCATGCTATCATCTACGATGTGGCTAAGATGGTGC
GA
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP
CAGAAATGGCCAGATTACTATGTTCCTACTGAGTTTAAAGGTATCGACAAGGAATTG
CCC
YRW
CAGAAATGGCCAGATTACTATGTTCCTACTGAGTTTAAAGGTATCGACAAGGAATTG
CCC
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP
ATAGTGTCTTTTTCATCAAAGAGGCTTACGGATATGGGGTTTGAATTCAAATACACTC
TG
YRW
ATAGTGTCTTTTTCATCAAAGAGGCTTACGGATATGGGGTTTGAATTCAAATACACTC
TG
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP
GAGGATATGTATCAAGGGGCCATTGAGACTTGCCGACAGAAGCAGTTGCTTCCCTTT
TCT
YRW
GAGGATATGTATCAAGGGGCCATTGAGACTTGCCGACAGAAGCAGTTGCTTCCCTTT
TCT
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP
ACCCGAAGCACTCTAGACAATGGAGAAGATAAAGAAACCATTCCCATATCTACTGAA
AAC
YRW
ACCCGAAGCACTCTAGACAATGGAGAAGATAAAGAAACCATTCCCATATCTACTGAA
AAC
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP
TATGCAAGTGGCAAGGAGAATGCACCAGTTGCAAATAGTACAGGACAGTTGACCAAT
GGT
YRW
TATGCAAGTGGCAAGGAGAATGCACCAGTTGCAAATAGTACAGGACAGTTGACCAAT
GGT
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
YLP GAAATCTAG
YRW GAAATCTAG
＊＊＊＊＊＊＊＊＊
YLP紫色茄子二氢黄酮醇4-还原酶核苷酸序列
YRW白色茄子二氢黄酮醇4-还原酶核苷酸序列
步骤二、白色茄子SmDFR基因的表达与拷贝数分析
1.YRW SmDFR基因的荧光定量PCR分析
取圆白茄的绿色茎，叶片，白色花瓣，未成熟果实白色果皮，商品成熟期果实的白色果皮，生理成熟期果实黄色果皮的总RNA，采用SmDFR-F2和SmDFR-R2引物对进行SmDFR基因的荧光定量PCR分析：
SmDFR-F2为上游引物，引物序列为5’-AAGGCTGTCAGGGAGTATTTCA-3’，
SmDFR-R2为下游引物，引物序列为5’-AGTTTCTGGTTCTCTTGGACATC-3’。
以SmACTIN基因作为内参对照，采用SmACTIN-F1和SmACTIN-R1引物对：
SmACTIN-F1为上游引物，引物序列为5’-TTCCTTGTATGCTAGTGGTCGTACAA-3’，
SmACTIN-R1为下游引物，引物序列为
5’-CTCAGCACCAATGGTAATAACTTGTCC-3’。
标准曲线采用克隆到pMD-19T载体上的DFR部分片段和ACTIN部分片段的质粒DNA绘制，设置5个标准样品，分别是质粒DNA原液和质粒DNA稀释10倍，100倍，1000倍和10000倍的DNA样品。定量PCR的操作采用PrimeScript RT-PCR Kit(Takara)，实验步骤按照试剂盒说明书进行。荧光定量PCR反应在Mastercycler ep realplex(Eppendorf)PCR仪上进行。反应条件为预变性94℃2min；94℃20s，52℃20s，72℃30s，35个循环；72℃1min。
实验结果表明，SmDFR在圆白茄的所有被测组织中的表达量都很低。同时与紫长茄相比，紫长茄的非紫色组织和圆白茄的对应组织的SmDFR的表达量基本一致，而紫长茄的紫色组织与圆白茄对应组织中，SmDFR紫色组织表达量要显著高于圆白茄的非紫色对应组织。
2.YRW mDFR基因的拷贝数分析
为了确定YRW SmDFR基因的拷贝数，提取圆白茄的基因组DNA，进行Southern杂交试验。提取DNA的具体方法如下：
取圆白茄的幼嫩叶片，采用CTAB方法提取。具体步骤如下：
(1)取0.3g新鲜幼嫩的叶片在液氮中快速研磨成粉末后，置于2ml的离心管中；加入500μlCTAB提取缓冲液，60℃水浴45min；CTAB提取缓冲液的配方为：CTAB 4g，氯化钠16.4g，Tris-HCl 20ml(pH 8.0)，0.5M EDTA 8ml，用去离子水70ml溶解后，定容到200ml，高压灭菌，冷却后加入0.5％的巯基乙醇(v/v)。
(2)待冷却后，加入500μl苯酚-氯仿-异戊醇(分析纯液体，25∶24∶1体积比)抽提混合液，混匀后，12000r/m离心10min；
(3)上清液转入新管，加入500μl氯仿-异戊醇(分析纯液体，24∶1体积比)抽提混合液，混匀后，12000r/m离心10min；取上清液转入新管，加入500ul冷处理的异丙醇，轻轻混匀，冰浴30min，4℃，8000r/m离心10min；
(4)倒去上清液，加入去离子水300ul溶解后，加入150ul，7.5mol/L NH4Ac混匀，冰浴20min，4℃，10000r/m离心20min；
(5)取上清液，加入预冷的无水乙醇900ul，冰浴20min，4℃，8000r/m离心20min；倒去上清液，70％乙醇冲洗两次，干燥后，加入去离子水150ul溶解后，加入RNase除去RNA，37℃水浴30min。DNA提取后，在分光光度计上测定DNA含量。
DNA探针采用回收纯化的SmDFR的cDNA的扩增片段，片段回收方法为将扩增SmDFR的cDNA全长序列电泳，紫外灯下割取DNA条带，用AxyPrepTM DNA gel Extraction Kit试剂盒回收，回收方法参照试剂盒说明书。使用DIG-High Prime DNA Labeling and DetectionStarter Kit I(Roche)试剂盒进行探针标记。标记方法参见试剂盒说明书。
采用
BamH I内切酶(Fermentas)进行基因组DNA的酶切。酶切体系参照内切酶说明书，在37℃水浴条件下，酶切6h。酶切后的DNA在20V电压下进行0.8％浓度琼脂糖电泳10h。电泳完毕后的琼脂糖凝胶采用进行脱嘌呤，变性和中和操作。将凝胶浸入100ml脱嘌呤液(0.25N的盐酸)中，15min，然后浸入100ml变性液(0.5mol/LNaOH，1.5mol/L氯化钠)30min，再浸入100ml中和液(0.5mol/L Tris-HCl，pH7.5，1.5mol/L氯化钠)30min。
凝胶浸入20×SSC液体中，采用VACUGENE XL BLOTTING UNIT真空转印系统(GE)将DNA转移到HYBOND-N+尼龙膜上。转印后的尼龙膜用2×SSC洗涤片刻后，120℃的烘箱内烘烤30min进行DNA交联。探针杂交和显色步骤也按照DIG-High Prime DNA Labeling andDetection Starter Kit I试剂盒说明书进行。20×SSC溶液配方为：氯化钠175.3g，柠檬酸钠88.2g用离子水定容到1L，pH7.0。2×SSC溶液为20×SSC溶液用去离子水稀释10倍。
DNA印迹结果显示，SmDFR在圆白茄中为单拷贝基因。
步骤三、白色茄子SmDFR酶的功能验证
1.YRW SmDFR原核表达载体的构建
YRW SmDFR基因的原核表达采用了pMAL-c2x载体(New England BioLabs)。载体采用BamHI和SalI双酶切，采用BamH I内切酶(Fermentas)进行基酶切操作。酶切体系参照内切酶说明书，在37℃水浴条件下，酶切15min。
DFR的ORF区域采用带有BamHI酶切位点的5’端引物和带有XhoI酶切位点的3’端引物扩增，引物序列为上游引物：5’-CGCGGATCCATGGCAAGTGAAGCTC-3’，下游引物：5’-ATTCTCGAGCTAGATTTCACCATTGGTCAA-3’。PCR扩增YRW SmDFR的ORF区域，每个循环内72℃延伸时间为90s，退火温度为60℃。扩增条带用BamHI和XhoI双酶切，酶切采用
BamH I内切酶(Fermentas)进行基酶切操作。酶切体系参照内切酶说明书，在37℃水浴条件下，酶切15min。回收步骤将扩增片段电泳，紫外灯下割取DNA条带，用AxyPrepTM DNA gel Extraction Kit试剂盒回收，回收方法参照试剂盒说明书。
酶切过的DNA和载体采用DNA Ligation Kit Ver.2.0试剂盒(Takara)连接，连接方法参见试剂盒说明书。热击转化入大肠杆菌BL-21感受态菌株。在有氨苄的LB固体平板培养基上37℃培养过夜，挑取单菌落，测序确认连接状况。LB培养基的配方为：配制每升培养基，用950ml去离子水中加入10g胰化蛋白胨，5g酵母提取物，10g氯化钠。调节pH至7.0，定容到1L体积。15psi高压下蒸汽灭菌20min。LB固体培养基配方为在LB液体培养基中，加入15g琼脂粉。15psi高压下蒸汽灭菌20min。
2.YRW SmDFR的融合蛋白诱导
挑选生长状态良好的BL-21菌株单克隆，转接到1L的LB液体培养基的三角瓶内，其中含有氨苄50μg/ml。37℃，200-225rpm摇菌4h左右，使OD600为1.0左右，将培养物转入20-22℃的摇床中以200-250rpm振摇1小时后加1ml 1M IPTG(异丙基-β-D-硫代吡喃半乳糖苷)(终浓度为1mM)，在20℃条件下以80-100rpm振摇过夜。随后10000rpm，4℃，离心10分钟后，收集大肠杆菌细胞。将沉淀物重悬于50ml裂解溶液中，并在冰上冷却10分钟。在冰浴条件下，使用超声细胞破碎仪破碎细胞。随后将破速的细胞在15000rpm条件下离心30min，取上清。在上清中加入1ml用裂解溶液清洗并平衡的直链淀粉微珠(Amylose resin)(NewEngland Biolabs)，同时再向溶液加入另50ml裂解溶液并平均分为两部分，4℃孵育过夜。之后1000rpm，4℃条件下慢速离心1min后，弃上清。用50ml清洗溶液清洗微珠三次后，用1ml洗脱溶液洗脱。
其中菌体裂解溶液的配方为：20mM Tris-HCl，pH8.0，200mM氯化钠，1mM PMSF，EDTA-free的蛋白酶抑制剂(Promega)，10％甘油；清洗溶液的配方为：20mM Tris-HCl，pH8.0，200mM氯化钠，10％甘油；洗脱溶液的配方为：20mM Tris-HCl，pH8.0，200mM氯化钠，50mM麦芽糖，10％甘油。
结果表明，YRW SmDFR的融合蛋白对于三种反应底物均具有活性。能够将二氢山奈酚，二氢檞皮素，二氢杨梅素转化为相应的底物。具有二氢黄酮醇4-还原酶的活性。并且对于二氢杨梅素的活性要显著大于二氢山奈酚和二氢檞皮素。但是在对于二氢杨梅素的活性上，YRWSmDFR的活性要明显低于YLP SmDFR。
Claims (4)
1.一种茄子花青素合成二氢黄酮醇4-还原酶的基因序列，其特征在于，该序列具有SEQID NO.1中从核苷酸第1-1149位所示的核苷酸序列。
2.根据权利要求1所述的茄子花青素合成二氢黄酮醇4-还原酶的基因序列，其特征是，所述的序列编码的多肽具有SEQ ID NO.2所示的氨基酸序列。
3.一种茄子花青素合成二氢黄酮醇4-还原酶的基因序列，其特征在于，该序列具有SEQID NO.3中从核苷酸第1-1149位所示的核苷酸序列。
4.根据权利要求3所述的茄子花青素合成二氢黄酮醇4-还原酶的基因序列，其特征是，所述的序列编码的多肽具有SEQ ID NO.4所示的氨基酸序列。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103215275A (zh) * | 2013-05-20 | 2013-07-24 | 上海交通大学 | 茄子花青素合成相关转录因子SmMYB的基因序列 |
| CN110229928A (zh) * | 2019-06-27 | 2019-09-13 | 江西省农业科学院蔬菜花卉研究所 | 用于茄子种质资源鉴定的分子标记组合及其应用 |
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| 《植物学报》 20031231 YANG GUO-HUA et al Molecular Cloning and characterization of a DFR from Developing Seeds of Blue-grained Wheat in Anthoctanin Biosynthetic Pathway 全文 1-4 第45卷, 第11期 * |
| 《生物技术通讯》 20090530 李春雷等 植物茄子花青素合成二氢黄酮醇4-还原酶的基因的研究进展 全文 1-4 第20卷, 第3期 * |
| 《茶叶科学》 20021231 王日为等 茶叶中花青素类物质研究进展 全文 1-4 , 第4期 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103215275A (zh) * | 2013-05-20 | 2013-07-24 | 上海交通大学 | 茄子花青素合成相关转录因子SmMYB的基因序列 |
| CN110229928A (zh) * | 2019-06-27 | 2019-09-13 | 江西省农业科学院蔬菜花卉研究所 | 用于茄子种质资源鉴定的分子标记组合及其应用 |
| CN110229928B (zh) * | 2019-06-27 | 2023-03-24 | 江西省农业科学院蔬菜花卉研究所 | 用于茄子种质资源鉴定的分子标记组合及其应用 |
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